Lymphocyte Development and Antigen Receptor Gene Rearrangement Part I Flashcards
What are the stages of lymphocyte maturation?
Stem cell->pro-lymphocyte->pre-lymphocyte->immature lymphocyte->mature lymphocyte
What are the major events during the stem cell and pro-lymphocyte stages of maturation?
Growth factor mediated commitment
Proliferation
initiation of antigen receptor gene rearrangment
What are the major events during pre-lymphocyte stage of maturation?
Selection of cells that express pre-antigen receptors
What are the major events during immature lymphocyte stage of maturation?
Selection of repertoire and acquisition of functional competence
What does the maturation of B and T lymphocytes involve?
Involves a series of events that occur in the generative lymphoid organs
The events of maturation include:
- commitment of progenitor cells to the B or T lymphoid lineage
- Proliferation of progenitors and immature (providing a large pool of cells for generation of lymphocytes)
- Sequential and ordered rearrangement of Ag receptors genes and the expression of antigen receptor proteins
- Selection events
- Differentiation of B and T cells into functionally and phenotypically distinct subpopulations
What does the selection events eliminate?
Potentially dangerous self-reactive cells
What do pluripotent stem cells give rise to?
Distinct B and T lineages
What gives rise to common lymphoid progenitor (CLP)?
Hematopoietic stem cells (HSCs)
What do CLPs give rise to?
B cells, T cells, and NK cells
What is the commitment to different lineages driven by?
By various transcription factors
What can Pro-B cells eventually differentiate into?
Follicular (FO) B cells
Marginal zone (MZ) B cells
B-1 cells
What can Pro-T cells commit either to?
αβ T cells or γδ T cells
What does the commitment to the B or T lineage depend on?
Depends on sequential signaling from several cell surface receptors
What does signaling activate?
Activates transcription factors that contribute to the commitment via induction of gene expression and rearrangements of Ag receptor gene
To develop B cells, what locus opens up?
The Ig heavy chain locus opens up and becomes accessible to the proteins that will mediate Ig gene rearrangement and expression
To develop αβ T cells, what locus opens up?
The TCR β gene locus opens up and becomes accessible for TCR gene rearrangement and expression
What transcription factors commit developing lymphocytes to the T cell lineage?
Notch-1 and GATA-3
What are the Notch family of proteins?
They are cell surface molecules that are proteolytically cleaved when they interact with specific ligands on neighboring cells
Where does the cleaved intracellular portion of Notch proteins migrate to?
To the nucleus and modulate the expression of specific target genes
Notch-1 together with GATA-3 induces what?
The expression of genes involved in development of αβ T cells
What do some of the genes expressed by Notch-1 and GATA-3 encode?
Some encode components of the pre-TCR and undergo V(D)J recombination
What TFs induce the expression of genes required for B cell development?
EBF, E2A, and Pax-5 transcription factors
What genes required for B cell development encode?
The Rag-1 and Rag-2 proteins (BCR rearrangement)
Surrogate light chains (pre-B cell receptor)
The Igα and Igβ proteins (the B cell receptor)
What stimulates the proliferation of the committed T and B cell progenitors?
Cytokines
What does proliferation ensure?
Ensures that a large pool of progenitor cells is available for generation of a high diversity of mature lymphocytes
If a pre-Ag receptor is successfully rearranged, what does it provide?
Provides survival signals that select the cell
IL-7 produced by stromal cells in the thymus drives what?
The proliferation of human T cell progenitors
What results in X-linked severe combined immunodeficiency disease (XSCID)
Mutations in the common γ chain, a protein that is shared by the type I cytokine receptors for several cytokines including IL-2, IL-7, and IL-15.
This blocks T cell and NK cell development, reflecting the requirement for IL-7 in T cell development and IL-15 for NK cells in humans
What cytokine is required for the development of NK cells?
IL-15
The mechanisms that makes genes available or unavailable in chromatin are considered to be what?
epigenetic mechanisms
How does DNA exist?
In chromosomes tightly bound to histones and non-histone proteins, forming chromatin
How may chromatin exist?
As relatively loosely packed euchromatin in which genes are available and are transcribed
Or very tightly packed heterochromatin in which genes are maintained in a silenced state
What does the structural organization of portions of chomosomes make some genes available for?
Transcription factors to initiate the transcription
What generally silences genes?
DNA methylation on cytosine residues
Histone post-translational modifications of the tails of nucleosomes render genes what?
Either active or inactive
The silencing of gene expression is done by what?
RNA-based mechanisms - by non-coding RNAs
What renders genes either active or inactive?
Post-translational acetylation, methylation, or ubiquitination modifications of the histone tails of nucleosomes
What can either enhance or suppress gene expression?
Active remodeling of chromatin by proteins called remodeling complexes
What are miRNAs?
microRNAs are a class of small noncoding RNAs (~22 nucleotides) that control gene expression at the post-transcriptional level by impairing translation or by promoting degradation of the target messenger RNA (mRNA)
What are required for recruitment of proteins that mediate gene recombination to form function Ag receptor genes?
Histone modifications in Ag receptor gene loci
CD4 and CD8 lineage commitment during T cell development depends on what?
epigenetic mechanisms (chromosome modifications) that silences the expression of the CD4 gene (heterochromatin state) in CD8 T cells
What do miRNA modulate during T cell development?
Gene expression
What results in a preferential loos of regulatory T cells and the consequent development of an autoimmune phenotype?
The deletion of Dicer, a key enzyme in miRNA generation
What results in the loss of Dicer in the B lineage?
A block at the pro-B to pre-B cell transition, primarily by being permissive for the apoptosis of pre-B cells
What have gene ablation studies revealed?
That other specific miRNAs are also involved in other steps in both B and T cell development
What plays a key role in preventing apoptosis of pre-B cells by inhibiting the expression of Bim?
miR17-92
What is Bim?
a pro-apoptotic Bcl-2 family protein
What targets a Notch receptor that plays important roles in proliferation, differentiation, and survival of T cells?
miR-150
How do individuals inherit alleles for L and H chains?
They codominantly inherit maternal and paternal sets of alleles for L and H chains
How many VLCL and VHCH alleles is expressed in a single B cell?
Only one - either from maternal or paternal
Having only one of the VLCL and VHCH alleles expressed in a single B cells is termed what?
This restriction is termed allelic exclusion
What does allelic exclusion also govern?
The expression of TCR cells
There are many B cells and T cells, therefore:
The maternal and paternal allotypes are expressed equally
Stem cells contain what?
Germline Ig and TCR
What are the variable regions of the chains in T and B cells determined by?
rearrangement of the DNA
What are the 3 mechanisms of DNA rearrangement?
Somatic recombination
mRNA splicing
Junctional diversity
The expression of BCR and TCR is initiated by what?
somatic recombination
What is the major mechanism of epitope-specific diversity of BCR and TCR?
DNA chromosomal rearrangment
What does DNA chromosomal rearrangement process include?
The deletion of DNA/RNA nucleotides and reannealing gene segments
What has 2 chromosomes encoding VH and VL chains from maternal and paternal?
A signel B cell
What part of the H and L chains are highly variable in amino acid sequence?
NH-terminus
All circulating immunoglobulins of the same isotype have what?
limited variability of constant (C) region
An individual B cell has many but all ______ BCRs
identical
How many unlinked gene groups encode immunoglobulins?
3
one for μ H-chains (chromosome 14), one for k chains (chromosome 2), and one for λ (chomosome 22) chains each on a different chromosome
Within each of the genes the encodes for Ig, there are what?
Multiple coding regions (exons) which recombine at the level of DNA to yield a binding site
How many variable segements are in each portion of Ig?
Heavy chain: 45
k: 35
λ: 30
How many variable segments are in each portion of TCR αβ?
α: 45
β: 50
How many diversity segments are in each portion of Ig?
Heavy Chain: 23
k: 0
λ: 0
How many diversity segments are in each portion of TCR αβ
α: 0
β: 2
What is the total potential repertoire with junctional diversity in Ig heavy chain and k?
~10^11
What is the total potential repertoire with junctional diversity in TCR αβ chains?
~10^16
What do recombination-activating genes 1 and 2 encode? (RAG)
Enzymes performing recombination of BCR and TCR during the process of VDJ recombination
The cellular expression of RAG1 and RAG2 is restricted to what?
B and T lymphocytes during their developmental stages
Each chromosome (maternal and paternal) encoding the components of BCR is trying to be what?
The first in genes’ rearrangement
Heavy chain has how many separate gene segments?
4 separate gene segments in chromosome 14: V, D, J, C
What does V, D, J, and C stand for?
V - variable
D - diversity
J - joining
C - constant
What does each segment for Heavy chain contain?
multiple copies of each segment
In a single B cell all copies but one are randomly deleted giving what?
A unique combination of V-D-J.
What does each B cell generate?
It’s own V-D-J sequence and thus all B cells are different
Immature B cell DNA has multiple copies, after recombination what does the mature B cell DNA have?
In mature B cell, each DNA segment is present as a single copy
What segment is first chosen in the immature B cell DNA and then whatever is between them is deleted?
First, D is chosen and DNA in between D and J is deleted. D and J are fused
D1—–D2———-J1—–J2
What segment is secondly chosen in immature B cell DNA?
V segment is chosen and DNA between V and DJ is deleted
What segment is thirdly chosen in immature B cell DNA?
J is chosen and DNA between VDJ and C is deleted
What does Cm and Cd stand for?
Cm for IgM and Cd for IgD
The chance to produce a “productive rearrangement” is:
~10%
What is productive rearrangement?
Rearrangement sequence made without stop codons in the sequence
What test will be done for selection of “productive rearrangement”?
A test including transcription and translation
When “productive rearrangement” is confirmed, what follows?
The “competition” is over and the recombination of other segments is STOPPED
How this is done remains UNKNOWN
If the VH rearrangement is productive then:
B cell proliferates for a while and “takes care” of the VL
What recombination rules do VL have?
The same as VH
There will be a second test for selection of “productive rearrangement”, this time for what sequence?
VL
What is the final result of B cell DNA rearrangement?
Each B cell produces only one kind of VH and VL
Because the number of combinations possible is very big, B cells:
Produced can recognize any Ag possible
How many nucleotides (N) long each segment of the H chains is?
V segments = 45
D segments =23
J segments = 6
C segments = 9
In a developing B cell, how is the functional VDJ gene produced for the H chain?
One of the V segments recombines with one of many D segments, which has already recombined with one of several J segments
In each B cell, what happens to the VDJ gene?
It is transcribed, spliced and translated into a H-chain protein
What does V-D-J recombination brings what close to the enhancer (enh)?
Brings promoter sequences (shown as P) close to the enhancer (enh).
What does the enhancer promote?
Promotes transcription of the rearranged V gene
Where does the C region of H chain B cell DNA lie?
Downstream of the rearranged V(D)J exon separated by the germline J-C intron
The rearranged B cell gene is transcribed to form what?
A primary (nuclear) RNA transcript
What does subsequent RNA splicing bring together?
The leader exon, the V(D)J exon, and the C region
What can the B cell mRNA be now translated on after splicing?
Translated on ribosomes to produce one of the chains of the antigen receptor
What segments make up the k L chain Germ line DNA?
35x V segments
5x J segments
1x C segment
What segments make up the λ L chain germ line DNA?
30x V segments
1x J segment
4x C segments
The germline sequence alpha-chain for TCR has what segments?
V segment has about 70-80 copies
J segment has 61 copies
The germline sequence beta-chain for TCR has what segments?
V segment has 52 copies
J segment has 13 (6+7) copies
Which TCR chain locus is on chromosome 7?
β
Which TCR chain locus is on chromosome 14?
α
Which TCR chain locus have D segments?
β
Where is junctional diversity generated?
At the points between the joining genes
What does junctional diversity result from?
The loss of nucleotides through the action of exonuclease(s) and from the addition of N and P nucleotides
Where are P nucleotides derived from?
From the asymmetric opening of hairpin loops
What does the opening of the hairpin loops produce?
Short, self-complementary single stranded extensions that can be incorporated into junctions, or may alternatively be removed via exonuclease activity
What leads to P nucleotides’ palindromic appearance and to their name?
The self-complementarity of P nucleotides
What are the steps of junctional diversity?
RAG cleaves the DNA hairpin at a random site
A single-stranded DNA end is formed
Depending on the site of cleavage, DNA may contain complementary nucleotides
Such stretches of nucleotides are called P nucleotides
What is the addition of N nucleotides aided by?
Terminal deoxynucleotidyl transferase (TdT)
What is the number of possible V-(D)-J combinations in Ig genes?
10^6
What is the number of possible V-(D)-J combinations?
3x10^6
During lymphocyte development, what do cell go through?
numerous checkpoints at which the developing cells are tested and continue to mature only if a preceding step in the process has been successfully completed
When is checkpoint #1?
After the production of the first polypeptide chaings of the two-chain Ag receptor is completed
When is checkpoint #2?
It follows the production of the second polypeptide chain of the Ag receptor once completed
What do checkpoints ensure?
That only lymphocytes that have successfully completed Ag receptor gene rearrangement processes are selected to mature
What does selection eliminate?
Potentially harmful self-reactive lymphocytes
What do Pre-Ag receptors and Ag receptors deliver during development?
Survival signals for their proliferation and continued maturation
What are pre-Ag receptors called in B cells and T cells? What do they contain?
pre-BCRs and pre-TCRs
They contain only ONE of the two polypeptide chains present in a mature Ag receptor
What do pre-BCRs contain?
The Ig μ heavy chain
What do pre-TCRs contain?
The TCR β chain
What do B or T cells must undergo?
Ag receptor gene rearrangements
What is the first Ag receptor gene to be completely rearranged in B cells?
The Ig heavy chain (IgH)
Cells that successfully rearrange their Ig heavy chain genes express what?
The μ heavy chain protein and assemble the pre-BCR
Developing T cells that make a ________ synthesize the TCR β chain protein and assemble the pre-TCR
productive TCR β chain gene arrangement
About ___% of developing B and T cells make productive rearrangements of Ag receptor gene (in-frame rearrangements capable of generating a protein)?
30
If cells make out-of-frame rearrangements, what happens?
The pre-antigen receptors are not expressed, the cells DO NOT receive survival signals and undergo apoptosis
What do assembled pre-BCR and pre-TCR provide?
signals for survival, proliferation and further development of early B and T lineage cells
What B and T lymphocytes proceed to express genes encoding the second chain of the BCR or TCR?
Cells with functional pre-Ag receptors
If these cells make productive rearrangements of the 2nd chain what happens?
They express the complete Ag receptor while they are still immature
Which T cells will be eliminated by apoptosis?
Those that are potentially harmful because they strongly recognize self-Ags
What happens to harmful B cells that strongly recognize self Ags?
They are induced to rearrange their 2nd chain of Ag receptors again
Lymphocytes that express useful Ag receptors are:
Preserved by a process called positive selection
What does positive selection ensure in the T cell lineage?
Ensures the maturation of CD8 or CD4 T cells whose receptors recognize appropriate self MHC molecules
T cells positively selected by self MHC molecules in the THYMUS are able to what?
recognize foreign Ag displayed by the same self MHC molecules on APCs in peripheral tissues
When does negative selection occur?
After Ag receptors are first expressed on developing B and T cells
What does negative selection do to B and T cells?
It eliminates developing T cells or alters developing B cells whose Ag receptors bind strongly to self Ags present in the thymus or bone marrow
What is clonal deletion?
A phenomenon when developing T cells with high affinity for self antigens are eliminated by apoptosis
Strongly self-reactive immature B cells may be induced to do what?
Receptor editing: Make further Ig gene rearrangements and thus evade self-reactivity
If B cell receptor editing fails, what follows?
The self-reactive B cells die, which is also called clonal deletion
What is an important mechanism for maintaining the central tolerance to many self Ags?
Negative selection of immature lymphocytes
