Lecture 4 and 5 B Cell Development and Primary Ab Repertoire Flashcards
1
Q
A single B cell makes multiple kinds of antibody specificity. True or False
A
FALSE
- makes one type of antibody specificity
(one VH and one VL)
AND a single plasma cell makes only one the of antibody; 1 kind of H chain and 1 kind of L chain
2
Q
What violates the rule that a single B cell makes only one type of antibody specificity (1 VH and 1 VL) or that a plasma cell makes only one type of Antibody? (1 H chain and 1 L chain)
A
B cells! violate the rule and synthesize two or more heavy chain isotopes simultaneously for the cell surface
IgM and IgD
3
Q
What mechanism facilitates the rule that individual B cells only make one antibody?
A
Allelic Exclusion
4
Q
Describe the following for Early Pro-B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - DJ **rearranging
- L chain Genes - Germline
- Surface Ig - ABSENT
5
Q
Describe the following for Late Pro-B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - V-DJ rearranging
- L chain Genes - germline
- Surface Ig - ABSENT
6
Q
Describe the following for Large Pre-B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - VDJ rearranged
- L chain Genes - germline
- Surface Ig - U chain at surface as part of pre-B cell receptor (intracellular)
7
Q
Describe the following for Small PRE-B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - VDJ rearranged
- L chain Genes - V-J rearranging
- Surface Ig - Intracellular U chain (meu)
8
Q
Describe the following for Immature B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - VDJ rearranged
- L chain Genes - VJ rearranged
- Surface Ig - IgM expressed on the cell surface
9
Q
Describe the following for Mature B cell:
- H chain genes
- L chain Genes
- Surface Ig
A
- H chain genes - VDJ rearranged
- L chain Genes - VJ rearranged
- Surface Ig - IgD and Ig M made from alternatively splicing H chain transcripts
10
Q
The cell that contains U-heavy chain of variable and Constant regions is called what? Where is it found only?
A
Pre-B Lymphocyte (has meu)
- found only in hematopoietic tissue such as bone marrow & fetal liver
11
Q
When are K or lambda light chains produced?
A
Immature B cells
12
Q
What is unique about immature B cells?
A
- do not proliferate and differentiate in response to antigens
- once a B cell expresses a complete heavy or light chain, it cannot produce another heavy or light chain containing a different V region
13
Q
Which type of B cells acquire a complete Ig and thus ANTIGEN SPECIFICITY?
Where can these be found?
A
Mature B Cells
- migrate out of bone marrow and can be found in PERIPHERAL CIRCULATION and lymphoid tissue
- continue to mature without antigenic stimulation
14
Q
What is the result of Mature B cells that co-express meu and delta heavy chains in association with the original kappa and lambda light chain?
A
Produce both membrane IgM and IgD
15
Q
Although mature B cells produce both membrane IgM and IgD, they still follow the rule that a single B cell makes only ONE TYPE of antigen specificity.
HOW?
A
They both contain the SAME V REGION! and thus the same antigen specificity
16
Q
What is allelic exclusion?
A
Only one IgH and one IgL allele are productively rearranged
- thus each B cell clone and its progeny are specific for only ONE ANTIGENIC DETERMINENT
17
Q
How does allelic exclusion occur?
A
Only one functional heavy chain VDJ and one functional VJ rearrangement occur in each cell
- only one allele is expression in a cell!
18
Q
What mediates the recombination of V, D, and J segments specifically?
A
RSS
- specific DNA RECOGNITION SEQUENCES located in the intervening DNA 3’ of each V exon and 5’ of each J segment
(downstream of V and upstream of J)
19
Q
What is an RSS?
A
Highly conserved stretched of seven or nine nucleotides separated by non-conserved 12 or 23 nucleotide spaces
20
Q
What recognizes the RSS, align, and cleave the DNA between the exon and the RSS?
A
RAG1 and RAG2
- two exon are ligated by DNA ligases
21
Q
What is a marker that B cells are developing in the bone marrow after bone marrow transplantation?
A
BREC!
- B cell recombination excision circles (PCR recognizes these)
22
Q
What is the mechanism by which secreted and membrane forms of U-Chain (meu) result?
A
ALTERNATIVE SPLICING!!!!
- either SC region in the primary transcript and DNa
or a C terminus transmembrane protein for IgM
23
Q
What is the same in IgM and IgD (membrane) in B cells?
A
VH & VL –> antigen specificity
24
Q
How can meu (u) and delta (d) chains with the same VH domains result on IgM and IgD B cells?
A
ALTERNATIVE SPLICING of primary transcripts (nuclear RNA)
25
During Gene rearrangement, which is the only residual segment that is NOT leftover both downstream and upstream?
D segment
- both upstream & downstream D is deleted (since D is in center of VDJ)
- upstream V and downstream J are left over
26
The first rearrangement in the heavy chain occurs where? (between what two segments)
Between D and J segment
27
Splicing of primary transcript to form the mRNA only occurs in the Heavy Chain Ig rearrangement.
True or False?
FALSE
- occurs in both
28
Where is Ig Gene Rearrangement occurring?
Is this antigen driven?
In the Bone Marrow (HSC stem cells undergo these changes)
NOT antigen driven & totally random
29
Describe the B cell development in Bone Marrow.
What type of cell does it start as and what type of cell does it end as? (7)
Name the key event that occurs at each step
1. HSC stem cell
2. Early Pro-B cell (D-J heavy)
3. Late Pro-B Cell (VDJ Heavy)
4. Large PRE-B cell (u chain on surface, but MAINLY intracellularly still)
5. Small PRE-B cell (V-J Light)
6. Immature B cell
(IgM expressed on cell surface!)
7. Mature B cell
- IgM and IgD expressed due to Alternative Splicing)
30
Where is mature B cell found?
in the PERIPHERAL blood
| - has IgM and IgD expressed on the surface
31
What are CD?
Cluster Differentiation
- CD are surface proteins/molecules on the surface of lymphoid cells
32
What does an immature B cell have on the surface of the cell?
IgM
33
Although IgM and IgD are expressed, what is the same that allows them to express the same antigen specificity?
VH and VL is the same for IgM and IgD
(variable chain is the same,
but heavy chain of the constant region is different)
34
What is the difference between V-J rearrangement in Light Chains and Heavy chains in terms of allele location?
V-J rearrangement can occur on both alleles!!!!
BUT VDJ rearrangement can only occur on 1 allele
- turns off rearrangement by putting a receptor on the surface
- sends a signal to shut OFF
35
When the large Pre-B cell binds to a pre-B cell receptor, what occurs?
REARRANGEMENT STOPS
- turns off heavy chain rearrangement once VDJ is complete on ONE allele
but expressing the U chain transiently at the surface as a B cell receptor
(V-J in light chain does not have this)
36
Once VDJ rearrangement is complete and a surface receptor for the Pre-B cell is formed, what gets turned on to start rearranging the light chain gene?
RAG
- V-J rearrangement occurring
- meu chain travels to the surface once V-J rearrangement has occurred
37
What serves as the B cell receptor in pre-B cells?
U chain being transiently expressed at the surface
38
What special signal is found in downstream V or upstream J?
RSS!
- highly conserved
- recognized by RAG
- splices the DNA & deletes the intron
39
What mediated the recombination process of Ig?
Recombination process is mediated by RAG!!!
and by the RSS which are downstream of V and upstream of J
40
How to Obtain Secreted IgM and Membrane IgM with the Same VDJ region? & How to Obtain IgM and IgD with the Same VDJ region?
Alternative Splicing
- secreted IgM and membrane IgM determined by alternative splicing (no DNA rearrangement at this point)
41
What is the mechanism by which Allelic Exclusion occurs?
What two things are required?
1. recognized by what DNA sequence
2. Enzyme that recognizes
1. RSS signal --> conserved heptamer/nonamer recognition sequences
2. RAG1/RAG2
- to recognize the RSS and splice in the appropriate region
Light chain: splice in lambda and kappa region of V downstream & J upstream
Heavy chain:
splice in V downstream
D upstream & downstream
J upstream
42
What are 4 mechanisms of Generation of Antibody Diversity?
1. Combinatorial V(D)J gene joining
2. N nucleotide addition by TdT(Terminal deoxynucleotidyl Transferase)
3. Combinatorial association of H and L chains
4. Somatic Hypermutation of Ig Genes – after B cells leave the bone marrow!!!!***
43
Describe Combinatorial Joining of V,D,J segments
Germline has multiple VH and VL genes
-Somatic recombination of Ig DNA allows for different V,D,J gene segments to associate
- leading to a large potential for generating different antibody specificities
- maximum number of possible combinations is the product of the number of V,(D),and J gene segments at each locus
- every clone of B cells and its progeny express a unique combo of V,D, and J genes
44
Describe Junctional diversity - N region addition.
1. What is added?
2. Where?
3. What enzyme mediates this?
1. Nucleotides, called N sequences (NOT PRESENT IN THE GERM LINE) can be added to-->
2. rearranged VDJ genes during rearrangement
3. Addition of new nucleotides is mediated by the enzyme TERMINAL DEOXYRIBONUCLEOTIDE TRANSFERASE (Td)
45
Describe the following:
Combinatorial association of
H and L chains
Combination of different H and L chain proteins contribute to diversity
How?
Because the V region of each chain participates in ANTIGEN RECOGNITION
-VH and VL can have a combinatorial joining, make one binding site together (multiply Vh*Vl = total number of binding sites
46
What are the results of the following:
B lineage cells in the bone marrow that have
1. Non-Functional V(D)J gene rearrangements
2. Express self-reactive antibody (3)
1. Non-Functional V(D)J gene rearrangements
= DELETED (apoptosis)
2. Express self-reactive antibody:
a) Deleted (apoptosis)
b) Anergic (non-responsive)
c) Receptor Editing
47
1. What enzyme is necessary for RECEPTOR EDITING of B cells that have anti-self reactivity?
2. Which specific area of the B cell immunoglobulin is changed?
3. Which area CANNOT be rearranged?
4. What will be changed in receptor editing?
1. RAG
2. only change the VJ light chain!!!
can only edit the LIGHT chain
3. No heavy chain VDJ rearrangement since this area was DELETED! (became part of the BREC)
4. Antibody binding site! (antigen specificity/Isotype)
48
How is the structure of TCR (T cell receptors) similar to BCR?
What is the binding site for a T cell?
1. Both have a structure that recognizes only ONE isotope
- consists of an Fab region and an Fc region
2. 2 chains: alpha & Beta
each has a variable region & a constant region
variable region of Alpha and variable region of Beta is the BINDING site
THUS each TCR can recognize only ONE ANTIGEN (similar to BCR)
49
Where does T cell recombination occur?
B cell?
Where do both initially form?
T cell = THYMUS
B cell = Bone Marrow
HSC stem cells initially form in the BONE marrow
50
True or false:
Allelic Exclusion only occurs in B cell receptors.
FALSE!
Occurs in both TCR's and BCR's
51
What is different about T gamma delta cells?
The polypeptide chains are designated gamma and delta instead of alpha and beta
TCR is a disulfide linked heterodimer of alpha and beta chains
52
What encodes the V regions of alpha and gamma chains?
The beta and delta?
V and J gene segments
VDJ gene segments
- TCR rearrange by the same mechanism as Ig genes
53
In T cells and B cells, only ONE VDJ and only ONE VJ rearrangement occurs.
True or False
TRUE!
Each T cell and B cell express a single TCR and BCR specific for one particular antigenic determinant
54
True or False:
Immunoglobulins are encoded by genes located on ONE chromosome
FALSE
- Ig are encoded by 3 gene families: for heavy chain, kappa, and lambda light chains
- the 3 gene families reside on separate chromosomes
55
With one immunoglobulin molecule, there can be 2 types of light chain.
True or False?
FALSE
- the light chain of a single antibody are IDENTICAL and the heavy chains are identical also
- Ig can either be lambda or kappa
56
Where does isotype switching occur?
What enzyme is important for this?
Which is rearranged:
- constant region
- variable region
Is antigen specificity changed?
1. PERIPHERY!
(Mature B cell)
2. needs the AID mediated cleavage in the Switch region
3. the looped DNA is cleaved out
new IgA or IgG3 produced as a result,
in the CONSTANT region of the heavy chain (variable region stays the same)
4. Antigen specificity is NOT changed, only the isotope
57
What are the only 2 DNA recombination mechanisms? DNA specifically!!!
1. V,D,J gene rearrangement
| 2. Isotype Switch
58
1. V,D,J gene rearrangement (2)
2. Isotype Switch (2)
State the following for these 2 DNA rearrangement mechanisms:
a) Signal
b) enzyme
c) location of recombination (periphery or bone marrow)
1.
a) RSS
b) enzyme RAG1/RAG2
c) BONE MARROW
2.
a) Switch Region
b) AID
c) OCCURS IN THE PERIPHERY
59
What do the following have in common:
CLASS SWITCH & Somatic Hypermutation
CLASS SWITCH & Somatic Hypermutation occurs in the PERIPHERY
60
How does N nucleotide addition by TdT create antibody diversity?
Changes the antibody binding site!
- amino acids that appear are changed since DNA at the end of V1 or in the J region is cleaved to make a BREC
* same as SOMATIC HYPERMUTATION --> mutation generates 1 base pair change
= different antigen specificity
61
Do T cells utilize RAG and RSS?
YES
- intervening DNA is deleted and we have a TREC
- also have receptor editing
62
What is the total diversity for T cells and B cells?
B cell = 10^13
| T cell = 10^18
63
T cells have somatic hypermutation & switch recombination (isotope switch)?
True or False?
FALSE
- once they are out of the thymus their specificity is NOT changed
only in B cells!!
T cells also DO NOT have a membrane and secreted form, this is only in B cells
64
What immunoglobulin is required for the primary response?
The secondary response?
IgM
- short lived, and comes down in a few weeks
- generates MEMORY during primary response
IgG
- class switch has occurred!
- no lag time
- larger, more effective response
65
What is the difference between V-J rearrangement in Light Chains and Heavy chains in terms of allele location?
FOR TCR and BCR
V-J rearrangement can occur on both alleles in TCR (only on 1 allele in BCR)
BUT VDJ rearrangement can only occur on 1 allele
- turns off rearrangement by putting a receptor on the surface
- sends a signal to shut OFF
