Lecture 5- Lymphocyte antigen receptor diversity Flashcards
What is somatic recombination?
A process in developing B cells and T cells where gene segments (Variable (V), Diversity (D), and Joining (J)) are rearranged to create diverse antigen receptor specificities. This occurs in the bone marrow for B cells and the thymus for T cells.
What is junctional diversity?
Additional diversity introduced at the junctions of recombined V(D)J gene segments due to the random addition (by terminal deoxynucleotidyl transferase, TdT) or deletion of nucleotides. This further increases the variability of antigen receptors beyond the combinatorial possibilities of somatic recombination. For B cells and T cells.
What is somatic hypermutation?
A process occurring in activated B cells within germinal centers, where point mutations are introduced into the variable regions of immunoglobulin (Ig) genes. This enhances antibody affinity through affinity maturation, helping to improve immune responses. For just B cells.
How does somatic hypermutation occur?
- Introduction of Mutations in the Variable Region
The enzyme Activation-Induced Cytidine Deaminase (AID) introduces point mutations (single nucleotide changes) specifically in the Variable (V) region of the immunoglobulin (Ig) gene.
These mutations alter the amino acids in the antigen-binding site of the antibody. - Generation of B Cell Variants
Because mutations occur randomly, some B cells produce antibodies with:
Higher affinity (stronger binding to the antigen)
Lower affinity (weaker binding)
No change in affinity (neutral effect) - Selection of High-Affinity B Cells
B cells compete for binding to antigens displayed by follicular dendritic cells (FDCs) in the germinal center.
Helper T cells provide survival signals to B cells with higher-affinity antibodies.
B cells with low or no affinity die by apoptosis. - Clonal Expansion of High-Affinity B Cells
The surviving high-affinity B cells proliferate and differentiate into:
Plasma cells (secrete high-affinity antibodies)
Memory B cells (for long-term immunity)
What is isotype switching?
A mechanism in B cells where the constant region of the antibody heavy chain is changed (e.g., IgM to IgG, IgA, or IgE), without altering the antigen-binding specificity. This allows different immunoglobulin classes to mediate distinct immune functions. For B cells.
How is a complete V domain generated?
Somatic recombination of separate gene segments. V domain of Ig heavy or light chain is encoded by more than one gene segment. Random selection of one of multiple gene segments gives rise to the great diversity of V domains.
What is the V domain encoded by?
2 separate gene segments: a V gene segment (first 95-101aa of V domain) and a joining or J gene segment (remaining 13 aa of V domain).
How are V and J segments joined?
Somatic recombination to create a continuous exon encoding the whole of the light chain V domain.
How is a complete Ig light chain mRNA made?
By joining the V region RNA to the C region sequence by RNA splicing.
Where are the genetic loci of Ig genes located?
2 light chain loci-chromosomes 22 and 2. 1 heavy light chain locus- chromosome 14.
What is the heavy chain encoded by?
3 separate gene segments: a V gene segment denoted VH. A joining or JH gene segment. A third gene segment called the diversity or DH gene segment which lies between the V and J segments.
What are the two separate stages of somatic recombination?
First: A D gene segment is joined to a J gene segment. Second: A VH gene segment is joined to the DJ segment to make a complete heavy chain V domain exon. Third a complete heavy chain mRNA is made by joining the transcribed V region RNA to the C region sequence by RNA splicing.
How does DNA rearrangement occur at the correct location relative to the V,D or J gene segment coding regions?
Guided by conserved non-coding sequences adjacent to the points where recombination takes place.
What are the 2 conserved blocks of nucleotides?
Heptamer- 5’ CACAGTG 3’ (always adjacent to the coding sequence). Nonamer 5’ ACAAAAACC 3’
What is the recombination signal sequence?
A Heptamer (7 nucleotides, contiguous with the coding sequence, the cleavage site where gene is cut)
A Spacer (12 or 23 nucleotides, non-coding)
A Nonamer (9 nucleotides, further from the coding sequence recruits RAG proteins)
What does the spacer do?
Acts as a buffer between the heptamer and nonamer.
Enforces the 12/23 rule, which ensures that only compatible gene segments recombine.
The 12/23 Rule:
A 12-nucleotide spacer can only pair with a 23-nucleotide spacer during recombination.
Prevents incorrect recombination, such as two V segments joining together instead of a V and a J.
How many joining events need to occur to form light and heavy chains?
One for a light chain and two for a heavy chain.
What is the molecular mechanism behind recombination?
Conserved spacer length corresponds to one or two turns of the DNA double helix which brings the heptamer and nonamer to the same side of the DNA helix. V(D)J recombinase can bind to the conserved sequences. The recombinase contains RAG-1 and RAG-2 subunits (recombination activating genes) which are lymphoid specific.
When does V(D)J recombination occur?
During initial development of primary lymphoid organs: Bone marrow (B cells) or thymus (T cells).
What are the enzymatic steps in RAG-dependent V(D)J rearrangement?
The DNA is in its original (germline) configuration, with separate V (Variable) and J (Joining) gene segments.
These segments need to be rearranged to form a functional antigen receptor.
RAG1/2 Binds RSS (Recombination Signal Sequences):
The RAG1/2 complex (Recombination Activating Genes) binds to the RSS (Recombination Signal Sequences) flanking the V and J gene segments.
RSS sequences guide the recombination machinery to the correct locations.
Synapsis of RAG Complexes:
The RAG1/2 complexes bring together the two RSS sites, forming a synaptic complex where both the V and J gene segments are aligned for cleavage.
Cleavage of RSSs:
The RAG1/2 complex introduces double-stranded breaks at the RSS sites.
This creates two types of DNA ends:
Coding Ends: Covalently closed hairpin loops that contain the gene segments.
Signal Ends: Blunt phosphorylated ends containing the RSS.
Coding Joints:
The hairpin loops are processed by the DNA repair machinery (Ku70/Ku80 complex) to generate coding joints, where V and J gene segments are ligated together.
Signal Joints:
The RSS signal ends are also joined by the Ku70/Ku80 complex, forming signal joints with phosphorylated blunt ends.
What happens to the coding joints?
They are modified further for more diversity using junctional diversity. * Ku70/Ku80 Binds DNA Ends: The Ku70/Ku80 complex binds the DNA hairpin ends to protect them and recruit repair proteins.
* DNA-PK:Artemis Opens Hairpin: The hairpin-shaped DNA ends are opened by Artemis (a nuclease) in complex with DNA-PK (DNA-dependent protein kinase).
○ Artemis can open the hairpin at various points, which adds more variability by generating P-nucleotides (palindromic sequences that add more randomness)
* TdT Processes DNA Ends: Terminal deoxynucleotidyl transferase (TdT) randomly adds nucleotides (N-nucleotides) to the DNA ends, increasing junctional diversity. Up to 20 not from the genome just completely random.
DNA Ligase IV:XRCC4 Ligates DNA Ends: The final step is joining the processed DNA ends together using DNA Ligase IV and XRCC4, creating the final coding joint. Any leftover unpaired nucleotides are removed by exonuclease.
What happens to the signal joints?
They only need to be processed not further modified. The signal joints are the blunt-ended RSS sequences that are simply joined together. Ku70/Ku80 Binds DNA Ends: The Ku70/Ku80 complex binds the signal ends. DNA Ligase IV:XRCC4 Ligates DNA Ends: The signal ends are directly ligated without further processing, forming a precise signal joint.
Where are the complementary determining regions encoded?
CDR1 and CDR2 encoded in the V segment. CDR3 encoded in the joint between V and J segments (light chains) and partly by the D segment (heavy chains).
When does somatic hypermutation occur?
After functional Ig genes have been assembled and in response to antigen and signals from activated T cells.
