B cell activation & organization of Immunoglobulin genes Flashcards
Overview of B cell development
List the following from most immature to most mature
( Pro - B cell, Mature B cell, Activated B cell, Lymphoid cell, Immature B cell, Pre - B cell, Hematopoietic stem cell )
Hematopoietic stem cell > Lymphoid cell > Pro - B cell > Pre - B cell > Immature B cell > Mature B cell > Activated B cell
Organization and Expression of Immunoglobulin genes
What does V, D, J, C stands for?
V: Variable
D: Diversity
J: Joining
C: Constant
Organization and Expression of Immunoglobulin genes
What is the structural difference between light - chain & heavy - chain?
Light chain : V - J arrangement
Heavy chain : V - D - J arrangement
Organization and Expression of Immunoglobulin genes
What are the two light chain gene?
Kappa & Lambda
Organization and Expression of Immunoglobulin genes
What is the order in rearranged Kappa - chain DNA?
( Intron x2 , Joined VJ gene segment, constant region, Leader exon )
Order :
Leader > Intron > Joined VJ gene segment > Intron > Constant region
Organization and Expression of Immunoglobulin genes
Which sequence in light chain is cleaved in RER?
Leader sequance
Organization and Expression of Immunoglobulin genes
Which of the following determines the class of antibodies?
(V, D, J, C)
C : Constant
Organization and Expression of Immunoglobulin genes
What are the difference in rearranged Kappa - chain DNA & rearranged Heavy - chain DNA?
Kappa :
-> Joined VJ gene segment
-> A constant region
Heavy :
-> Joined VDJ gene segment
-> A series of C gene segments
( For antibody class )
Organization and Expression of Immunoglobulin genes
What is the two class of antibodies that is expressed initially in primary RNA transcription?
C(mu) : IgM
C(delta) : IgD
Organization and Expression of Immunoglobulin genes
What is the process that determines whether IgM or IgD is produced?
RNA splicing
Organization and Expression of Immunoglobulin genes
Does a mature B cell that expresses both IgM & IgD have the same identical antigenic specificity, and why?
Yes
What are the 2 mechanisms of variable - region DNA rearrangements?
RSS ( Recombination signal sequences )
-> direct recombination
VDJ recombinases
-> joining gene segment
Organization and Expression of Immunoglobulin genes
What is the 2 types of RSSs? ( Recombination signal sequences )
One turn RSS
Two turn RSS
Organization and Expression of Immunoglobulin genes
What are the 2 -amers in RSS?
And whats the properties of it?
Heptamer
-> Palindromic
Nonamer
-> AT - rich
Organization and Expression of Immunoglobulin genes
What is the “ One - turn / two - turn joining rule “ about ?
One - turn RSS can only join with two - turn RSS
Organization and Expression of Immunoglobulin genes
What is the recombinase enzyme that can recognize RSSs?
RAG - 1 / 2
( Recombination - activating gene )
Organization and Expression of Immunoglobulin genes
What is the enzyme that cleave the hairpin structure and trim a few nucleotides from the coding sequence?
Endonuclease
Organization and Expression of Immunoglobulin genes
What is the enzyme that adds nucleotides to the cut ends of coding sequence of heavy chain?
TdT
( Terminal deoxynucleotidyl transferase )
Organization and Expression of Immunoglobulin genes
What is the enzyme that repair and ligate the coding sequences?
DSBR enzymes
( Double - strand break repair )
Does the RAG enzymes cleave both strand of the DNA at the junctures of the RSS and the coding sequence?
Nope, they cleave one strand of DNA
Organization and Expression of Immunoglobulin genes
What is P - addition and which 2 enzymes are involved?
Cleavage of hairpin by endonuclease
-> Sites for addition of P - nucleotides
- -> DSBR enzymes add complementary nucleotides
Organization and Expression of Immunoglobulin genes
What is N - addition and which 3 enzymes are involved?
Cleavage of hairpin by endonuclease
-> Sites for addition of P - nucleotides
- -> DSBR enzymes add complementary nucleotides
- - -> TdT adds N - nucleotides
Organization and Expression of Immunoglobulin genes
Does N - addition happens in both light chain and heavy chain?
Nope
It only happens in heavy chain
Organization and Expression of Immunoglobulin genes
What is junctional flexibility?
The selection of which nucleotide in two coding sequences (e.g. V & J / D & J) to be joined together can be completely random.
E.g. V ( ATG ) & J ( CGC )
-> ATGCGC
-> ATGGC
-> ACGC
-> ATCGC
etc
Organization and Expression of Immunoglobulin genes
What is the disadvantage of junctional flexibility?
It can produce stop codons which interrupt translation
Organization and Expression of Immunoglobulin genes
What are the 5 things that creates antibody diversity?
V(D)J rearrangement
P - region nucleotide addition
N - region nucleotide addition
Junctional flexibility
Somatic hypermutation
Organization and Expression of Immunoglobulin genes
Which of the following occurs in bone marrow / germinal center?
V(D)J rearrangement
P - region nucleotide addition
N - region nucleotide addition
Junctional flexibility
Somatic hypermutation
Bone marrow :
V(D)J rearrangement
P - region nucleotide addition
N - region nucleotide addition
Junctional flexibility
Germinal center :
Somatic hypermutation
Organization and Expression of Immunoglobulin genes
What is the mutation rate in somatic hypermutation? 10 ^ (??) / bp / mutation
10 ^ (-3) / bp / mutation
Organization and Expression of Immunoglobulin genes
Somatic hypermutation guarantees at least one mutation per every (??) division
Two
Organization and Expression of Immunoglobulin genes
What is allelic exclusion?
Allelic exclusion ensures single antigenic specificity
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what is first produced in progenitor B cell?
D and J component of VDJ heavy chain
Organization and Expression of Immunoglobulin genes
In allelic exclusion, which is the first allele checked?
Mu heavy chain (Mu allele #1)
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what does Mu heavy chain (Mu allele #1) inhibits & induces?
Inhibits rearrangement of the Mu allele #2
Induces Kappa rearrangement
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what is the second allele checked after Mu heavy chain (Mu allele #1) is productive?
Kappa light chain (Kappa allele #1)
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what happens when both Mu heavy chain (Mu allele #1) & Kappa light chain (Kappa allele #1) are productive alleles?
Antibodies are successfully produced
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what does Mu heavy chain (Mu allele #1) & Kappa light chain (Kappa allele #1) inhibits & induces?
Inhibits
-> rearrangement of the Kappa allele #1
-> Lambda rearrangement
Induces nothing
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what happens if Kappa light chain (Kappa allele #1) is nonproductive?
Kappa light chain (Kappa allele #2) is checked
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what if both Kappa light chain (Kappa allele #1 & #2) are nonproductive?
Lambda light chain ( Lambda allele #1) is checked
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what does Mu heavy chain (Mu allele #1) & Lambda light chain (Lambda allele #1) inhibits & induces?
Inhibits rearrangement of Lambda allele #2
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what happens if both Mu allele are nonproductive?
Apoptosis
Organization and Expression of Immunoglobulin genes
In allelic exclusion, what happens if both Kappa allele & Lambda allele are nonproductive?
Apoptosis
Organization and Expression of Immunoglobulin genes
Which two class of membrane bound antibodies are simultaneously expressed in mature naïve B cell?
mIgM & mIgD
Organization and Expression of Immunoglobulin genes
What are the difference between sIgM & mIgM?
sIgM
-> Contains hydrophilic portion that are encoded by S exon of C(mu)
mIgM
-> Contains both hydrophilic & hydrophobic portion that are encoded by M1 & M2 exons of C(mu)
- -> Hydrophilic : Outside + Cytoplasm
- -> Hydrophobic : Membrane
Organization and Expression of Immunoglobulin genes
What processing action in RNA transcription causes the difference between sIgM & mIgM?
RNA splicing
Organization and Expression of Immunoglobulin genes
Which of the two organelles is essential for sIg production?
RER & Golgi
Organization and Expression of Immunoglobulin genes
What are the 4 things that happen in RER for sIg production?
Translation of heavy chain & light chain
Cleavage of Leader sequence
Assembly of heavy chain & light chain to form monomer
Assembly of monomers to form homodimer
Organization and Expression of Immunoglobulin genes
What are the 2 things that happen in Golgi for sIg production?
Post translational modification of homodimer
-> Addition of oligosaccharide
Packaged by secretory vesicles
Organization and Expression of Immunoglobulin genes
What is the difference in Secretion between sIg & mIg
Same thing happens in RER & Golgi
However
-> Hydrophobic part of mIg becomes embedded on the membrane of secretory vesicle
-> Fusion of secretory vesicle with cell membrane causes mIg to stay on the membrane instead of being released
Organization and Expression of Immunoglobulin genes
What are the 3 types of regulatory elements in Ig genes?
Promoters
Enhancers
Silencers
Organization and Expression of Immunoglobulin genes
Where can promoters be found and what does it do?
200 bp upstream from transcription initiation site
Promotes initiation of RNA transcription in a specific direction
Organization and Expression of Immunoglobulin genes
Where can enhancers be found and what does it do?
upstream / downstream of genes ( in this case VDJ )
It can stimulate promoter to initiate transcription regardless its position / direction
Organization and Expression of Immunoglobulin genes
Where can silencer be found and what does it do?
upstream or downstream of genes
( in this case VDJ )
It can inhibit / repress gene transcription regardless its position / direction
Organization and Expression of Immunoglobulin genes
Does each V ( heavy chain ) & V ( light chain ) segment has a promoter & leader sequence?
Yes
Organization and Expression of Immunoglobulin genes
Which of the following DNA does not contain silencers?
Heavy chain DNA
Kappa chain DNA
Lambda chain DNA
Lambda chain DNA
Organization and Expression of Immunoglobulin genes
Explain the mechanism in class switching ( from IgM -> IgA ), Including the following words
Switch region
Constant exon region
Switch recombinase
In C gene after VDJ genes
There are switch region right before every different constant exon region
(e.g. S(IgM) C(IgM) S (IgD) C(IgD) )
When B cell desires to switch IgM class to IgA
Switch recombinase aids in forming a loop from S(IgM) -> C(IgE) )
( According to order IgM/D/G/E/A )
Removal of the loop brings S(IgA) in proximity to VDJ genes, allowing transcription of VDJ C(IgA)
Organization and Expression of Immunoglobulin genes
What is the switch factor (cytokine)?
IL - 4
B - cell Maturation, Activation & Differentiation
How long can plasma cell survive?
1 - 2 weeks
B - cell Maturation, Activation & Differentiation
What is the function of IL - 7?
It down regulates the adhesion molecules
B - cell Maturation, Activation & Differentiation
What triggers the expression of IL - 7 receptor on Pre - B cells?
Binding of c-Kit -> SCF
*c-Kit -> Bone marrow stromal cell
SCF -> Pro - B cell
B - cell Maturation, Activation & Differentiation
What is the protein that help transduce activating signals of mIg on B cell ?
Ig - alpha / Ig - beta heterodimers
B - cell Maturation, Activation & Differentiation
What are the two signals that drive B cells into & through the cell cycle?
Ag - mIg
CD40L
B - cell Maturation, Activation & Differentiation
What does Ag - mIg induce?
Expression of MHC - 2 & B7 on B cell
B - cell Maturation, Activation & Differentiation
What is the purpose of expressing MHC - 2 on B cell?
Recognization of MHC - 2 by Th cell activates Th cell
B - cell Maturation, Activation & Differentiation
What is the purpose of expressing B7 on B cell?
Allow B7 - CD28 interactions which co - stimulate Th cell with MHC - 2 recognition
B - cell Maturation, Activation & Differentiation
What happens when Th cell is activated by the co - simulation?
Th cell begins to express CD40L
-> Interaction between CD40 & CD40L (2)
B - cell Maturation, Activation & Differentiation
What happens after both signals is present & active?
B cells express cytokines receptor
Th cell release cytokines
-> IL - 2
-> IL - 4
-> IL - 5
Signals supports progression of B cell
-> DNA synthesis
-> Differentiation
B - cell Maturation, Activation & Differentiation
Which of the following cytokine induce class switch to
IgG2a / IgG3?
IL - 2
IL - 4
IL - 5
IFN - gamma
TGF - beta
IFN - gamma
B - cell Maturation, Activation & Differentiation
Which of the following cytokine induce class switch to
IgA / IgG2b?
IL - 2
IL - 4
IL - 5
IFN - gamma
TGF - beta
TGF - beta
B - cell Maturation, Activation & Differentiation
Which of the following cytokine induce class switch to
IgE / IgG1?
IL - 2
IL - 4
IL - 5
IFN - gamma
TGF - beta
IL - 4
B - cell Maturation, Activation & Differentiation
Which of the following cytokine induce class switch to
IgM?
IL - 2
IL - 4
IL - 5
IFN - gamma
TGF - beta
IL - 2
IL - 4
IL - 5
Match the following
IFN - gamma
TGF - beta Th1
IL - 4
IL - 2 Th2
IL - 5
Th1
-> IFN - gamma
-> TGF - beta
Th2
IL - 2
IL - 4
IL - 5
