Lecture 10 - Lymphocyte Development and Antigen Receptor Gene Rearrangement Flashcards
What are the stages of lymphocyte maturation?
L10 S4
Commitment:
-progenitor cells commit to B or T lymphoid lineage
Proliferation:
-expansion of immature lymphocyte population
Rearrangement:
-rearrangement of Ag receptor genes to produce BCR/TCR
Selection:
-eliminate self-reactivate cells
Differentiation:
-differentiation of pro-B/T lymphocytes into different subclassifcations
What can pro-B and T cells differentiate into?
L10 S5
Pro-B cells:
- follicular (FO)
- marginal zone (MZ)
- B-1 cells
Pro-T cells:
- αβ T cells (~90%)
- γδ T cells (~10%)
What is the function of IL-7 in lymphocyte development and what stimulates its release?
L10 S6
If a pre-Ag receptor is successfully rearranged it provides a survival signal (?)
IL-7 is produced by stromal cells in both bone marrow and the thymus and stimulates proliferation and development of B cells and T cells respectively
What transcription factors are responsible for T cell and B cell development and Ag receptor rearrangement?
L10 S8-9
T cell:
- Notch-1
- GATA-3: genes involved in αβ T cells
- genes expressed: pre-TCR, Rag-1, and Rag-2
B cell:
- EBF (early B cell factor)
- E2A
- Pax-5
- genes expressed: Rag-1, Rag-2, surrogate light chain (pre-BCR), and Igα/β
What are the different gene products that make of Igs?
How does the term “allelic exclusion” relate to these gene products?
L10 S15-16
Gene products:
- μ H-chain
- κ L-chain
- λ L-chain
Allelic exclusion:
- in each B-cell, only either the maternal or paternal copy of the H-chain and only one of the 4 L-chains is used to generate the BCR
- this generates different allotypes
Something similar occurs in T cells
What mechanisms are responsible for Ag receptor diversity?
L10 S17
Chromosomal rearrangement
V(D)J recombination:
-Rag1/2 initiates DS-DNA breaks and repairs via NHEJ
What is the structural basis of BCR H-chain diverstiy?
L10 S18;20;25
The heavy chain of the BCR is made of 4 different segments, each of which have several different copies:
- V (variable) (45 copies)
- D (diversity) (23 copies)
- J (joining) (6 copies)
- C (constant) (10 copies)
During development, all but one copy of V, D, and J is deleted creating a unique V-D-J combination for that B cell.
This occurs through VDJ recombination using the Rag-1/2 genes
What is the process by which V(D)J recombination occurs?
L10 S23
- first, one copy of D and J are selected then all DNA between them is deleted
- second, one copy of V is selected then all DNA between it and DJ is deleted
- third, C is selected and all DNA between it and VDJ is deleted
This occurs in both parental and maternal DNA (competition).
There is a ~10% chance that the VDJ sequence is viable (does not contain a premature stop codon). Combination is tested and if productive, recombination stops.
What is the structural basis of BCR L-chain diverstiy? When does generation of this segment occur?
L10 S18;24;25
The light chain of the BCR is made of 3 different segments, each of which have several different copies:
- V (variable) (35 copies)
- D (not found in L-chain)
- J (joining) (5 copies)
- C (constant) (2 copies; κ or λ)
After generation of productive H-chain and a period of proliferation, L-chain is generated by deletion of all but one copies of V and J
This occurs through VJ recombination using the Rag-1/2 genes
How is it determined if the BCR will be IgD or IgM?
L10 S26
Alternate splicing of C portion of heavy chain during BCR production incorporates either the Cμ copy or Cδ copy generating IgM or IgD respectively in the naïve B cell.
How does isotype switching occur?
L10 S26
Class-switch recombination (CSR):
- AID (activation-induced cytidine deaminase; exclusive to activated B cells) introduces uracil to switch (S) regions of DNA causing breaks
- DNA is repaired and Cμ/Cδ segment is replaced with Cγ, Cε, or Cα to produce IgG, IgE, or IgA respectively
What is the structural basis of TCR α-chain diverstiy? When does generation of this segment occur.
L10 S28-29
The α-chain of the TCR is made of 3 different segments, some of which have several different copies:
- V (variable) (45 copies)
- D (not found in α-chain)
- J (joining) (50 copies)
- C (constant)
After generation of functional β-chain, α-chain is generated by deletion of all but one copies of V and J.
This occurs through VJ recombination using the Rag-1/2 genes
What is the structural basis of TCR β-chain diverstiy?
L10 S28-29
The β chain of the BCR is made of 4 different segments, each of which have several different copies:
- V (variable) (50 copies)
- D (diversity) (2 copies)
- J (joining) (4 copies)
- C (constant) (2 copies)
During development, all but one copy of V, D, and J is deleted creating a unique V-D-J combination for that T cell.
This occurs through VDJ recombination using the Rag-1/2 genes
What is junctional diversity and what is responsible for it?
L10 S30-34
Junctional diversity is the addition of P and N nucleotides between V(D)J segments when they are combined.
Rag-1/2 asymmetrically cleaves hairpin loops at the end of each section and adds P nucleotides to even the cleaved ends.
Terminal deoxynucleotidyl transferase (TdT) add N nucleotides in the gap between the segments.
This massively increases the diversity of TCRs (10^6 -> 10^16) and BCRs (10^6 -> 10^11)
What occurs at each of the two checkpoints in the selection process for lymphocytes?
L10 S40-46
First checkpoint:
- pre-BCR consisting of heavy chain and surrogate light chain or pre-TCR consisting of β-chain and surrogate chain
- if pre-receptor is produced from in-frame rearrangements, cell receives survival signal (~30%)
- if no pre-receptor is produced due to out-of-frame rearrangements cell undergoes apoptosis
Second checkpoint (positive selection):
- TCR consisting of β-chain and α-chain or BCR consisting of heavy chain and light chain
- if TCR/BCR recognize MHC molecules but not self antigens, they develop into mature T/B-cells
Second checkpoint (negative selection):
- TCR consisting of β-chain and α-chain or BCR consisting of heavy chain and light chain (κ)
- if TCR is self-reacting, the T cell is eliminated via clonal deletion
- if BCR is self-reacting the B cell undergoes receptor editing using light chain (λ); if still self reactive, B cell is eliminated via clonal deletion
