Generation of B cell & Antibody Diversity Flashcards
What are antibodies?
➝ Y shaped molecules expressed by immune cells to target pathogens
What is the function of antibodies?
➝ Prevent bacteria entering cells
➝ neutralise toxins
What are the components of antibodies?
➝ two heavy chains and two light chains
How many classes do the heavy chains have and what are they?
➝ 5 classes
➝𝝻,δ,γ,ɑ,ε
What are the subclasses of the heavy chains?
➝γ1, γ2, γ3, γ4
➝ɑ1, ɑ2
How many domains is the heavy chain made of?
➝ 4 domains
➝ 1 variable region and 3 constant regions
How many domains is the light chain made of?
➝ 2 domains
➝ 1 variable region and 1 constant regino
What region is the same for antibodies of the same class?
➝ The constant regions
What is the constant region responsible for?
➝ biological activities
What does the variable region bind to?
➝ specific epitopes of specific pathogens
What are the two forms of antibodies?
➝ Secreted
➝ membrane bound
What is the final form of antibodies?
➝ secreted
During development where are the antibodies found?
➝ During development it is anchored to the plasma membrane of B cells
What are membrane bound antibodies also called?
➝ B cell receptors
What is the function of the antibody as a receptor?
➝ to sense changes in the environment
What can antibodies form when they are secreted?
➝ pentamers
What can B cell development be split into?
➝ Antigen independent stage in the bone marrow
➝ Antigen dependent stage in the blood and lymph node
Describe how plasma and memory B cells are formed?
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
What are changes at the DNA level called?
➝ Somatic recombination
What are 4 examples of somatic recombination?
➝ VDJ recombination
➝ Tdt nucleotide addition
➝ somatic hypermutation
➝ class switching
What are changes at the mRNA level called?
➝ Differential splicing
What are two examples of differential splicing?
➝ IgM and IgD
➝ membrane bound and secreted Ig
What is each chain made of in the antibody?
➝ one polypeptide
What is the structure of a polypeptide in the antibody?
➝ NH3 region at the N terminus
➝ the hinge region between CH1 and CH2
What is the hinge region of the antibody for?
➝ THis makes the antibody flex so the active site will exposed
What is attached to the CH2 region and makes part of the active site?
➝ Glycan group
What are receptors of the immune system attracted to?
➝ glycosylation pocket
What is the Fv (variable fragment) made from?
➝ variable region of the light and heavy chains working together
What is the Fab (antibody fragment) made from?
➝ THe variable fragment + first constant domain
What is the Fc made from?
➝ heavy chain CH2 and CH3
What is the CDR?
➝ complementarity determining regions
➝ three finger like protrusions in the variable region
What do the CDRs do and where are they found?
➝ they interact with the antigens
➝ they are in the variable light and heavy regions
How many resting B cells does the body make?
➝ 1,000,000,000 and they each contain a unique B cell receptor
When does the B cell undergo DNA recombination?
➝ At the Pro-B stage
Describe the antigen independent phase
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
When does the pre-B cell become a mature B cell?
➝ once it expresses IgM and IgD
What are the three genetic loci encoding Ig?
➝ two for light chain kappa and lambda locus
➝ one for a heavy chain
On what chromosome is the light chain lamba located on?
➝ 22
On what chromosome is the kappa light chain located on?
➝ 2
on what chromosome is the heavy chain located on?
➝ 14
How many antibody genes are inherited?
➝ none
➝ no complete genes are inherited only gene segments
What generates Ig sequences?
➝ arranging the gene segments in different combination
What are the regions in the light chain?
➝ V, J and C
What are the regions in the heavy chain?
➝ V - variable
➝ D - diversity
➝ J - joining
➝ C𝝻 - constant
What do the V,D,J etc. regions code for?
➝ once they are rearranged at the DNA level they will code for the CDR and variable region
What codes for CDR3?
➝ J or D/J region codes for CDR3
What is the most variable region of the Ab?
➝ CDR3
What are the 3 components of the kappa light chain?
➝ 40 variable segments
➝ 5 joining segments
➝ constant region segments
Describe how the kappa chain is transcribed and made?
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
What are the 4 components of the gamma heavy chain?
➝ 51 variable V segments
➝ 27 diversity D segments
➝ 6 joining J segments
➝ Constant region segments
Describe how the gamma heavy chain is synthesized?
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
What is combinatorial diversity?
➝ finding the number of possibilites available
What are the 7 ways that antibody diversity is generated?
➝ 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
What is an RSS?
➝ Recombination signal sequences - conserved sequences upstream or downstream of gene segments
What do ‘turns’ in RSS consist of?
➝ a heptamer and a nonamer with 12 or 23bp spacer
Describe the one turn/two turn rule (12/23 rule)
➝ 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
Describe how the V2 and J1 segments are recombined in the light chain?
➝ 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
What enzymes form the hairpin?
➝ RAG1 and RAG2
Describe how P and N nucleotides are added?
➝ 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
What does junctional flexibility mean?
➝ The coding joints lose base pairs
What is an advantage and a disadvantage of junctional diversity?
➝ Good = antibody diversity
➝ Bad = non-productive rearrangements (incorrect reading frame) - wasteful process
How many copies of Ig gene do we have?
➝ two
➝ one maternal and one paternal
How many Ig genes are expressed?
➝ 2 in most cases
How many alleles are expressed for heavy and light chains?
➝ 1 of each
What is the order of rearrangement of antibody chains?
➝ heavy > kappa > lambda
