Generation of B cell & Antibody Diversity

Question 1

What are antibodies?

Answer 1

➝ Y shaped molecules expressed by immune cells to target pathogens

Question 2

What is the function of antibodies?

Answer 2

➝ Prevent bacteria entering cells

➝ neutralise toxins

Question 3

What are the components of antibodies?

Answer 3

➝ two heavy chains and two light chains

Question 4

How many classes do the heavy chains have and what are they?

Answer 4

➝ 5 classes

➝𝝻,δ,γ,ɑ,ε

Question 5

What are the subclasses of the heavy chains?

Answer 5

➝γ1, γ2, γ3, γ4

➝ɑ1, ɑ2

Question 6

How many domains is the heavy chain made of?

Answer 6

➝ 4 domains

➝ 1 variable region and 3 constant regions

Question 7

How many domains is the light chain made of?

Answer 7

➝ 2 domains

➝ 1 variable region and 1 constant regino

Question 8

What region is the same for antibodies of the same class?

Answer 8

➝ The constant regions

Question 9

What is the constant region responsible for?

Answer 9

➝ biological activities

Question 10

What does the variable region bind to?

Answer 10

➝ specific epitopes of specific pathogens

Question 11

What are the two forms of antibodies?

Answer 11

➝ Secreted

➝ membrane bound

Question 12

What is the final form of antibodies?

Answer 12

➝ secreted

Question 13

During development where are the antibodies found?

Answer 13

➝ During development it is anchored to the plasma membrane of B cells

Question 14

What are membrane bound antibodies also called?

Answer 14

➝ B cell receptors

Question 15

What is the function of the antibody as a receptor?

Answer 15

➝ to sense changes in the environment

Question 16

What can antibodies form when they are secreted?

Answer 16

➝ pentamers

Question 17

What can B cell development be split into?

Answer 17

➝ Antigen independent stage in the bone marrow

➝ Antigen dependent stage in the blood and lymph node

Question 18

Describe how plasma and memory B cells are formed?

Answer 18

1) a stem cell in the bone marrow differentiates into a Pro-B cell
2) the pro-B cell undergoes the first of the DNA recombinations (V ➝ DJ) that codes in the heavy chain variable region
a) it becomes a pre-B cell once it makes a functional heavy chain
3) the pre-B cell undergoes another recombination (V ➝ J) to code in the light chain variable and constant regions in its DNA
a) It then becomes in an immature B cell
4) The B cell then continues to mature until it expresses both IgM and IgD through differential mRNA splicing
5) it then becomes a mature recirculating B cell (patrol blood and spleen)
6) the mature recirculating B cells are reactivated when the body encounters a pathogen
7) they migrate into the germinal center (GC) to hone its variable region to the specific pathogen
8) it undergoes affinity maturation and class switching then it differentiates into plasma cells which secrete antibodies and memory B cells

Question 19

What are changes at the DNA level called?

Answer 19

➝ Somatic recombination

Question 20

What are 4 examples of somatic recombination?

Answer 20

➝ VDJ recombination
➝ Tdt nucleotide addition
➝ somatic hypermutation
➝ class switching

Question 21

What are changes at the mRNA level called?

Answer 21

➝ Differential splicing

Question 22

What are two examples of differential splicing?

Answer 22

➝ IgM and IgD

➝ membrane bound and secreted Ig

Question 23

What is each chain made of in the antibody?

Answer 23

➝ one polypeptide

Question 24

What is the structure of a polypeptide in the antibody?

Answer 24

➝ NH3 region at the N terminus

➝ the hinge region between CH1 and CH2

Question 25

What is the hinge region of the antibody for?

Answer 25

➝ THis makes the antibody flex so the active site will exposed

Question 26

What is attached to the CH2 region and makes part of the active site?

Answer 26

➝ Glycan group

Question 27

What are receptors of the immune system attracted to?

Answer 27

➝ glycosylation pocket

Question 28

What is the Fv (variable fragment) made from?

Answer 28

➝ variable region of the light and heavy chains working together

Question 29

What is the Fab (antibody fragment) made from?

Answer 29

➝ THe variable fragment + first constant domain

Question 30

What is the Fc made from?

Answer 30

➝ heavy chain CH2 and CH3

Question 31

What is the CDR?

Answer 31

➝ complementarity determining regions | ➝ three finger like protrusions in the variable region

Question 32

What do the CDRs do and where are they found?

Answer 32

➝ they interact with the antigens | ➝ they are in the variable light and heavy regions

Question 33

How many resting B cells does the body make?

Answer 33

➝ 1,000,000,000 and they each contain a unique B cell receptor

Question 34

When does the B cell undergo DNA recombination?

Answer 34

➝ At the Pro-B stage

Question 35

Describe the antigen independent phase

Answer 35

1) the V region recombines with the DJ region that is already recombined 2) during this,junctional flexibility occurs which gives more diversity to the antibody and P and N nucleotide addition 3) this codes for the heavy chain variable region 4) this is co-expressed with the 𝝻 constant region 5) the 𝝻 constant region is for the IgM antibody class 6) the pre-B cells expresses a placeholder light chain because the light chain has not been generated yet 7) it undergoes a third recombination V➝ J coding for the light chain variable and constant region 8) The immature B cell expresses IgM

Question 36

When does the pre-B cell become a mature B cell?

Answer 36

➝ once it expresses IgM and IgD

Question 37

What are the three genetic loci encoding Ig?

Answer 37

➝ two for light chain kappa and lambda locus | ➝ one for a heavy chain

Question 38

On what chromosome is the light chain lamba located on?

Answer 38

➝ 22

Question 39

On what chromosome is the kappa light chain located on?

Answer 39

➝ 2

Question 40

on what chromosome is the heavy chain located on?

Answer 40

➝ 14

Question 41

How many antibody genes are inherited?

Answer 41

➝ none | ➝ no complete genes are inherited only gene segments

Question 42

What generates Ig sequences?

Answer 42

➝ arranging the gene segments in different combination

Question 43

What are the regions in the light chain?

Answer 43

➝ V, J and C

Question 44

What are the regions in the heavy chain?

Answer 44

➝ V - variable ➝ D - diversity ➝ J - joining ➝ C𝝻 - constant

Question 45

What do the V,D,J etc. regions code for?

Answer 45

➝ once they are rearranged at the DNA level they will code for the CDR and variable region

Question 46

What codes for CDR3?

Answer 46

➝ J or D/J region codes for CDR3

Question 47

What is the most variable region of the Ab?

Answer 47

➝ CDR3

Question 48

What are the 3 components of the kappa light chain?

Answer 48

➝ 40 variable segments ➝ 5 joining segments ➝ constant region segments

Question 49

Describe how the kappa chain is transcribed and made?

Answer 49

1) in front of each V segment there is a leader sequence 2) one of the segments from V1-V40 are selected at random 3) one J region is selected 4) at the DNA level this recombined so you have LVJC 5) this is transcribed to mRNA 6) in the primary RNA transcript the extra unneeded parts are spliced out such as the region between the leader and the variable region 7) the part between the J and the C segment is spliced out (the extra J segment) 8) this becomes mature mRNA with the stop codon and the poly-A-tail 9) this is translated to a polypeptide 10) once the kappa chain has correctly folded the leader sequence is cleaved off

Question 50

What are the 4 components of the gamma heavy chain?

Answer 50

➝ 51 variable V segments ➝ 27 diversity D segments ➝ 6 joining J segments ➝ Constant region segments

Question 51

Describe how the gamma heavy chain is synthesized?

Answer 51

1) A D and a J are selected to recombine 2) A V is selected to recombine with the D and the J 3) this is transcribed into the mRNA transcript, only the first two constant regions the C mu and the C delta will be transcribed with the rest of the LDVJ segments 4) The B cels can express IgM and IgD when they are mature 5) the B cell has a population of mRNA transcripts and some of them will be spliced to make a) LVDJ Cmu and it becomes a Mu heavy chain b) LVDJ and the J and the Cmu are removed and it becomes LVDJ C delta - delta heavy chain 6) by alternative splicing the B cell can make two different classes of B cell receptor without having to alter the DNA sequence

Question 52

What is combinatorial diversity?

Answer 52

➝ finding the number of possibilites available

Question 53

What are the 7 ways that antibody diversity is generated?

Answer 53

➝ Multiple germline V,D and J gene segments ➝ combination V-J and V-D-J joining ➝ Junctional flexbility ➝ P-nucleotide addition ➝ N-nucleotide addition ➝ Combinatorial association of heavy and light chains ➝ Somatic hypermutation during affinity maturation

Question 54

What is an RSS?

Answer 54

➝ Recombination signal sequences - conserved sequences upstream or downstream of gene segments

Question 55

What do 'turns' in RSS consist of?

Answer 55

➝ a heptamer and a nonamer with 12 or 23bp spacer

Question 56

Describe the one turn/two turn rule (12/23 rule)

Answer 56

➝ Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer heavy chain ➝ the two turn is located downstream of every V segment and upstream of every J segment ➝ the one turn is located upstream and downstream of the D segment ➝ this ensures that the correct parts recombine e.g D and J ➝ but J and J have only 12 so they do not recombine

Question 57

Describe how the V2 and J1 segments are recombined in the light chain?

Answer 57

➝ recombination only occurs between a 12bp spacer and a 23bp spacer ➝ RAG1/2 bind to the RSS sequence ➝ RAG1/2 bind to each other and form a hairpin with the sequences between the spacers ➝ KU70/KU80 form smaller hairpins with the loops remaining on the V2 and J1 segments

Question 58

What enzymes form the hairpin?

Answer 58

➝ RAG1 and RAG2

Question 59

Describe how P and N nucleotides are added?

Answer 59

➝ Overhangs are produced when artemis cuts the hairpin loops formed by KU70/80 on the ends of the segments ➝ repair enzymes add nucleotides to the overhangs ➝ the P nucleotides are the ones that fill the overhangs ➝ in the heavy chain there is another enzyme called TdT which adds the N nucleotides between the two segments that need to be joined

Question 60

What does junctional flexibility mean?

Answer 60

➝ The coding joints lose base pairs

Question 61

What is an advantage and a disadvantage of junctional diversity?

Answer 61

➝ Good = antibody diversity | ➝ Bad = non-productive rearrangements (incorrect reading frame) - wasteful process

Question 62

How many copies of Ig gene do we have?

Answer 62

➝ two | ➝ one maternal and one paternal

Question 63

How many Ig genes are expressed?

Answer 63

➝ 2 in most cases

Question 64

How many alleles are expressed for heavy and light chains?

Answer 64

➝ 1 of each

Question 65

What is the order of rearrangement of antibody chains?

Answer 65

➝ heavy > kappa > lambda