Lymphocyte Development and Antigen Receptor Gene Rearrangement Flashcards
The maturation of B and T lymphocytes involves a series of events that occur in the generative lymphoid organs. What are these events in order?
- Commitment of progenitor cells to the B lymphoid or T lymphoid lineage.
- Proliferation of progenitors and immature lymphocytes (providing a large pool of cells for generation of lymphocytes).
- Sequential and ordered rearrangement of Ag receptor genes and the expression of antigen receptor proteins.
- Selection events (eliminate potentially dangerous self-reactive cells).
- Differentiation of B and T cells into functionally and phenotypically distinct subpopulations.
What do pluripotent stem cells give rise to?
Distinct B and T lineasges
What type of cell does hematopoietic stem cells (HSCs) give rise to?
Common Lymphoid Progenitor (CLP)
Which three types of cells do CLPs give rise to?
- B cells
- T cells
- NK cells
What drives the commitment to different lineages of CLPs?
transcription factors
What cell types can Pro-B cells eventually differentiate into?
- Follicular (FO) B cells
- Marginal zone (MZ) B cells
- B-1 cells
What cell types can Pro-T cells commit to?
- alpha-beta T cells
- gamma-delta T cells
What stimulates the proliferation of the committed T and B cell progenitors?
cytokines
* IL-7 for T cell progenitors
* Unknown for B cell progenitors
Why is proliferation of B and T cell progenitors so important?
Proliferation ensures that a large pool of progenitor cells is available for generation of a high diversity of mature lymphocytes.
What happens if a pre-Ag receptor is successfully rearranged?
It provides survival signals that select the cell.
In the thymus, which cell type produces IL-7 to drive proliferation of human T cell progenitors?
Stromal Cells
What cytokine is the development of NK cells dependent on?
IL-15
What does the commitment to the B or T cell lineage depend on?
Sequential signaling from several cell surface receptors.
You know that the commitment to the B or T cell lineage is dependent on sequential signaling from several cell surface receptors. What does this signaling activate?
Activates transcription factors that contribute to the commitment via induction of gene expression and rearrangements of Ag receptor gene.
What are the two ways in which activated transcription factors contribute to the commitment of the B or T cell lineage?
- gene expression of Ag receptor gene
- rearrangements of Ag receptor gene
In developing B cells, what happens to the Ig heavy chain locus?
Ig heavy chain locus opens up and becomes accessible to the proteins that will mediate Ig gene rearrangement and expression.
In developing alpha-beta T cells, what happens to the TCR ß gene?
TCR ß 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
- GATA-3
What are Notch family of proteins?
Cell surface molecules that are proteolytically cleaved when they interact with specific ligands on neighboring cells.
How does the Motch protein modulate expression of specific target genes?
The cleaved intracellular portion of Notch protein migrates to the nucleus and modulates the expression of specific target genes.
Which T cell type does GATA3 induce the expressionf genes for?
alpha-beta T cells
True or False:
Some genes (which encode the components of pre-TCR) undergo V(D)J recombination.
True
Which transcription factors induce the expression of genes required for B cell development?
- EBF
- E2A
- Pax-5
Which proteins regulate BCR rearrangement?
- Rag-1
- Rag-2
What do EBF, E2A, and Pax-5 transcription factors include genes encoding for what?
- Rag-1 and Rag-2 proteins regulating the BCR rearrangement
- The surrogate light chains (pre-B cell receptor)
- The Ig-alpha and Ig-beta signaling proteins of the B cell receptor complex
What does DNA methylation on cytosine residues generally do?
Silences Genes
What do non-coding RNAs do to gene expression?
silence gene expression
What do epigenetic mechanisms do in lymphocyte development?
Make genes available or unavailable in chromatin.
True or False:
The structural organization of portions of chromosomes makes some genes available for transcription factors to initiate the transcription.
True
What is a class of small noncoding RNAs (22 NT) that control gene expression at the post-transcriptional level by impairing translation or by promoting degradation of the target messenger RNA (mRNA)?
microRNAs (miRs)
Does allelic exclusion govern the expression of TCR?
Yes
You know that individuals codoninantly inherit maternal and parental sets of alleles for L and H chains. But how is VLCL and VHCH alleles of the maternal or parental expressed in a single B cell?
ONLY one of the VLCL and VHCH alleles (either maternal or parental) is expressed in a single B cell.
Are maternal and parental allotpes expressed equally?
Yes - the maternal and parental allotypes are expressed equally since there are many B cells and T cells.
How are the variable regions of the chains in T and B cells determined?
By rearrangement of the DNA.
What is the major mechanism of epitope-specific diversity of BCR and TCR?
DNA chromosomal rearrangement.
What are the 3 different DNA chromosomal rearrangement mechanisms that determine epitope-specific diversity of BCR and TCR?
- somatic recombination
- mRNA splicing
- junctional diversity
** The process includes deletion of DNA/RNA nucleotides and reannealing gene segments. **
What do stem cells (a precursor of both T and B cells) contain?
- germline Ig
- TCR
True or False:
In achieving BCR diversity, each chromosome (maternal and parental) encoding the components of BCR is trying to be the first in genes’ rearrangement.
True
What are the 4 heavy chain separate gene segments on chromosome 14?
- “V” - variable
- “D” - diversity
- “J” - joining
- “C”- constant
Fill in the Blank:
In a single B cell all copies but _____ are randomly _____ giving a unique comination of __-__-__. Each B cell generates it’s own V-D-J sequence and thus all B cells are different.
one; deleted; V-D-J
What are Rag-1 and Rag-2 proteins?
The recombination-activating genes 1 and 2 which encode enzymes performing recombination of BCR and TCR during the process of VDJ recombination.
When is the cellular expression of RAG1 and RAG2 restricted?
The cellular expression of RAG1 and RAG2 is restricted to B and T lymphocytes during their developmental stages.
List the steps showing how BCR diversity is achieved.
- First, D and J are chosen and DNA in between them is deleted.
- Second, V segment is chosen and DNA between V and DJ is deleted.
- Next, J is chosen and DNA between VDJ and C is deleted (CM for IgM and CD for IgD).
- The chance to produce a “productive rearrangement” (without stop codons in the sequence) is about 10%.
- There will be a test (transcription + translation) for selection of “productive rearrangement”.
- When “productive rearrangement” is confirmed - the “competition” is over and the recombination of other segments is STOPPED.
- How it is done remains unknown.
If the VH rearrangement is productive, then what happens?
B cell proliferates for a while and “takes care” of the VL.
* there will be a second test for selection of “productive rearrangement”, this time for the VL chain *
* the recombination rules are the same for VL chain *
What is the final result after the first and second “productive rearragement” test have been performed?
Each B cell produces only one kind of VH and VL.
How are B cells able to recognize any Ag possible?
Because the number of combinations possible is very big, B cells produced can recognize any Ag possible.
In BCR diversity, does the light chain have any D segments?
NO
You know that the process of V(D)J recombination generates a repertoire of different TCR (3x106) and BCR (1x106) molecules for recognition of various microbial antigens and that this V(D)J recombination alone is not enough to produce a huge variety of TCRs and BCRs which would recognize all antigens possible. How is further specificity obtained?
JUNCTIONAL DIVERSITY further increases the diversity of TCRs (1x1016) and BCRs (1x1011).
Where is juctional diversity generated?
generated at the points between the joining genes
What does junctional diversity result from?
Results from THE LOSS of nucleotides through the action of exonuclease(s) and from THE ADDITION of N and P nucleotides.
What type of opening in the DNA generates P nucleotides?
An asymmetric opening of hairpin loops generates P nucleotides.
What is unique about P nucleotides that lead to their palindromic appearance and their name?
Self-complementarity of P nucleotides leads their palindromic appearance and to their name.
What does the opening of the hairpin loops produce?
Opening of the hairpin loops produces short, self-complementary single stranded extensions that can be incorporated into junctions, or can be removed via an exonuclease activity.
Which protein cleaves hairpin loops and adds P nucleotides?
RAG proteins
After RAG proteins have cleaves hairpin loops and adds P nucleotides, which protein adds nucleotides to connect the DNA?
Terminal Deoxynucleotidyl Transferase (TdT)
Why is the selection process important?
Selection process eliminates potentially harmful self-reactive lymphocytes.
What do the critical checkpoints in the selection process ensure?
Ensure that only LYMPHOCYTES that have sucessfully completed Ag receptor gene rearrangement processes are selected to mature.
During the selection process, when does checkpoint #1 occur?
After the production of the first polypeptide chain of the two-chain Ag receptor is completed.
During the selection process, when does checkpoint #2 occur?
Follows the production of the second polypeptide chain after the Ag receptor is completed.
True or False:
During lymphocyte development, the cells 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.
True
What chain does the pre-BCRs contain?
Ig µ heavy chain
What chain does the pre-TCRs contain?
TCR ß chain
What do pre-Ag and Ag receptors deliver for the proliferation and continued maturation for pre-BCRs and pre-TCRs?
survival signals
True or False:
The pre-Ag receptors contain only one polypeptide chain present in a mature Ag receptor.
True
What is the first Ig chain for the Ag receptor gene to be completely rearranged in B cells?
Ig Heavy Chain (IgH)
What is the next step after developing B cells successfully rearrange their Ig heavy chain genes?
Express the µ heavy chain protein and assemble the pre-BCR.
What is synthesized due to the developing T cells that make a productive TCR ß chain gene rearrangement?
The TCR ß chain protein and assemble the pre-TCR.
What percentage of developing B and T cells make productive in-frame rearrangements of Ag receptor gene (capable of generating a protein)?
30%
What happens if developing B and T cells make a out-of-frame rearrangement?
The pre-Ag receptors are not expressed, the cells do not receive survival signals and undergo apoptosis.
What do assembled pre-BCRs and pre-TCRs provide signals for?
- survival
- proliferation
- further development of early B and T lineage cells
B and T lymphocytes with what proceed to express genes encoding the second chain of the BCR or TCR?
FUNICTIONAL pre-Ag receptors
If B or T lymphocytes make productive rearrangements of the 2nd chain, do they express the complete Ag receptor while they are immature or mature?
immature
Through what process are lymphocytes that express useful Ag receptors preserved by?
Positive Selection
True or False:
T cells positively selected by self MHC molecules in the thymus are able to recognize foreign Ag displayed by the same self MHC molecules (class I MHC or class II MHC, respectively).
True
What is the difference between how potentially harmful T or B cells that strongly recognize self Ags are handled in positive selection?
- Harmful T cells that strongly recognize self Ags -> eliminated by apoptosis.
- Harmful B cells that strongly recognize self Ags -> induced to rearrange their 2nd chain of Ag receptors again.
How are lymphocytes that express useful Ag receptors are preserved by what process?
Positive Selection
What does positive selection of T cells ensure?
The maturation of CD8 or CD4 T cells whose receptors don’t recognize self-Ags but can recognize MHC molecules (class I MHC or class II MHC, respectively).
True or False:
T cell positively selected by self MHC molecules in the thymus are able to recognize foreign Ag displayed by the same self MHC molecules on APCs in peripheral tissues.
True
