L11 B cell development I Flashcards
what are b cells and what do they do?
- MATURE B cells express the B Cell Receptor - BCR
(membrane bound Immunoglobulin) on their surface. - The BCR recognises whole antigen (without the need for it
to be processed). - This activates the B cell (along with a second signal).
- Immunoglobulin genes are then diversified (class switching,
somatic mutation)
mature B cells have undergone recombination produce mature immunoglobulin gene. Naive b cells are mature but not yet exposed to antigen. Immunoglobulin genes can be diversified so specificity can be changed. - B cells then differentiate into:
– plasma cells that secrete ANTIBODIES (same
Immunoglobulin as BCR)
– long lived memory cells – Recall responses/vaccination - ANTIBODY binds whole pathogens and facilitates a range of
functions, depending on its Fc region (class/isotype).
immunoglobulin structure?
made up of two identical heavy chains and two identical light chain. heavy and light chain variable regions determine antigen binding. only the heavy chain determines effector functions of the fc portion. Immunoglobulin structure: fab fragment- the arms of the antibody where antigen binding is taking place.
Fc fragments: stem of the y shaped molecule giving antibodies its effector functions. Antibody: 2 identical copies of heavy chains consistuting the fc fragments (effector function) and have 2 identical copies of the light chain. Need identical to ensure they are all recognising the same thing.
variable regions are made up of 2 or 3 gene segments: VJ or VDJ.
both heavy and light chain have constant regions. The antigen binding region is encoded by different gene segments so V and Jin light chain and V(D)J in heavy chain. Variable diversity in joining. These fragents encode the ends of the arms, the last immunoglobulin folded domain that makes the structure of the antibody. But they crucially form complementarity determining regions- unstructured loops that are crucial for determining antigen binding.
how do b cells generate diversity to recognise diverse range of pathogens?
Generation of antibody diversity – V(D)J
Recombination
B cells generate diverse immunoglobulins by cutting and pasting gene segments together in the heavy chain and in the light chain.
Rearrangement of Immunoglobulin Genes
* Antigen specificity determined by CDR – Complementarity
determining regions
* CDR H3 formed by VDJ combination
* Light chains only have V and J segments
D - J recombination 1st
V - DJ recombination 2nd
The junction between V, D and J
segments determines antigen
specificity
In the case of the light chain there are 2 alleles: kappa and lamda. Recombination occurs in ordered manner and both have v d and j segments.
Framework regions are encoded by V segment
* They fold to form the domain structure of the
variable region
* CDRs are unstructured loops that determine
antigen binding specificity
* CDR 3 is encoded by the VDJ junction and
is most variable
*combinatorial diversity is an image have to look at it
what is combinatorial diversity?
Combinatorial diversity: first thing constituting diversity of immunoglobulins. Almost 8000 combinations of vdj and 200 + 140 vj. Any of those kappa and lambda light chains can be joined with a heavy chain so 2.6x10^4 possible combinations.
what mediates gene segment recombination?
The joining mechanism for recombining gene segments during V(D)J recombination is initiated by the enzymes RAG1 and RAG2 (Recombination Activating Genes 1 and 2). These proteins recognize recombination signal sequences (RSS) flanking the V, D, and J gene segments and bring the segments together to facilitate recombination.
This process involves:
Formation of hairpin loops:
The DNA is cut at the RSS, and the free ends of the DNA form hairpin loops to maintain genome integrity (to avoid open free DNA).
Opening the hairpins:
The hairpins are then opened at random points by enzymes, introducing junctional diversity. This imprecision at the site of joining results in diversity in the final recombined gene sequence.
Adding palindromic (P) nucleotides:
When the hairpins are opened, the DNA ends often form overhangs, creating a palindromic sequence (P nucleotides) as they fold back on themselves.
Adding or removing nucleotides:
To create a blunt end for joining, nucleotides may be removed from the overhangs, or additional random nucleotides (N nucleotides) are inserted into the gap by the enzyme Terminal deoxynucleotidyl transferase (TdT). N nucleotide addition is entirely random and significantly enhances diversity.
Imprecise joining:
The imprecise nature of this process—where hairpins are opened, and nucleotides are added or removed—creates further variability and is a key contributor to junctional diversity.
Combinatorial diversity:
This mechanism works alongside the random combination of V, D, and J segments (combinatorial diversity) to generate a vast repertoire of antibodies or T cell receptors.
easier explanation:
1. What is V(D)J recombination?
This is a process where B cells and T cells create a huge variety of antibodies (for B cells) or T-cell receptors (for T cells) to recognize many different pathogens.
It achieves this by rearranging specific segments of DNA called V (Variable), D (Diversity), and J (Joining) segments.
2. Role of RAG1 and RAG2
RAG1 and RAG2 are special enzymes that cut DNA at specific places called Recombination Signal Sequences (RSS) next to the V, D, and J segments.
These enzymes bring one V segment, one D segment, and one J segment close together for recombination.
3. Formation of Hairpin Loops
After RAG enzymes cut the DNA, the free ends of the DNA fold into hairpin loops.
Why? This helps protect the DNA from breaking apart completely.
4. Opening the Hairpins
Another enzyme comes in and opens the hairpins at random spots.
This is important because the randomness creates diversity in the resulting DNA sequence.
5. Adding Palindromic (P) Nucleotides
When the hairpins are opened, the ends of the DNA sometimes form small sequences that read the same forward and backward (like “GAAG” or “CAGC”).
These are called P nucleotides and are created naturally as the DNA folds back on itself.
6. Adding or Removing Extra Nucleotides
An enzyme called Terminal deoxynucleotidyl transferase (TdT) can:
Add random nucleotides (N nucleotides) to the open ends.
Remove existing nucleotides if needed to make the DNA ends blunt.
This randomness is called junctional diversity and is another way the immune system generates variety.
7. Joining the DNA
The DNA ends are then joined together to form a complete V-D-J segment.
The joining isn’t perfect—it’s imprecise, but this imprecision adds even more variety to the final antibody or receptor.
8. Combinatorial Diversity
On top of the randomness of junctional diversity, the immune system can mix and match different V, D, and J segments to create an enormous number of combinations.
b cell development and clonal manner?
B cell development and clonal manner: once b cells have rearranged ig genes and formed fully assembled immunoglobulin protein, it becomes mature b cell and is released into periphery as a naive b cell. Can circulate for years. Clonal expansion once detects antigen. Largely occurs in germinal centre. These individual clones can further diversify and differentiate into plasma cells and memory cells.
how does the recombination form cdrs and how many genes are there for the v d and j segments?
Rearrangement of immunoglobulin genes: germline configuration- immature b cells. Dont need to know exact number of genes but magnitude. 50 v genes 30 d 6 j Being selected and recombined with the d segment and intervening segments are lost. But crucially the bits upstream of that are still there. Only bits in between are lost from the genome.
Within v segments: framework regions making the physical immunoglobulin structures and within thema re CDRs- the unstructured loops at the ends and 2 of them are encoded by the v segments. Within 3rd cdr= formed by that junction of vdj being brought together.
Junction between VDJ sequence determines antigen specificity: the cd3 is important in determining what the final outcome is going to be.
how many recombination events are successful?
The recombined exon must
maintain its codon reading frame to make a
functional protein.
* Random insertions and deletions will destroy the
triplet codon reading frame in 2 out of 3 cases.
* So only 1 out of 3 recombination events are
“Successful”.
methods of diversity generation of immunoglobulin vs tcr?
gene rearrangement: both
heterodimer formation: heavy and light chain (x2) vs alpha with beta and gamma with delta?
hypermutation: only immunoglobulin
class switching: only immunoglobulin
recombination as b cells differentiate/develop in the bone marrow from hsc?
As B cells develop recombination
of the heavy and light chains
occurs in controlled order.
1st - Heavy chain D-J
2nd - Heavy chain V-DJ
3rd- Light chain V-J
bone marrow microenvironment?
stromal cells line the bone marrow
- mixture of endothelial, fibroblasts, fat cells
- provide growth signals to developing B cells
* Provide essential cell-cell contact
-VLA-4/VCAM-1 (CAMs)
- Kit/SCF
* AND soluble growth factors (cytokines)
- Interleukin 7
Differentiation of B cells in the bone marrow from HSCs:
Bone marrow microenvironment and cell-cell contact initiate the recombination process. IL-7 further drives the recombination process. Developing B cells are exposed to these signals in the bone marrow, and they control the processes involved in generating diversity, such as the rearrangement of the immunoglobulin genes
stages of B cell development: (1) HSC stem cell
HSC stem cell: H chain genes- germline (aka not rearranged)
L chain genes- germline
surface ig-absent
no RAG (for recombination)
no TdT (For recombination diversity)
no λ5 or VpreB (surrogate light chain)
stages of B cell development: (2) Early pro-B cell
H chain genes – when cells receive their early growth factors, there is activation and recombination of the heavy chain genes D to J rearrangement on both alleles.
V(D)J recombination starts with the D to J joining on both alleles.
After a successful rearrangement on one allele, allelic exclusion ensures that the second allele is not rearranged.
This prevents the B cell from producing two different heavy chains, ensuring it only recognizes one antigen with its receptor.
L chain genes – Germline
Surface Ig – Absent
RAG and TdT expressed (allow recombination)
No 5 or VpreB expression (surrogate light chain)
stages of B cell development: (3) Late pro-B cell
late-pro B cell: H chain genes- V to DJ
rearrangement
L chain genes – Germline
Surface Ig – Absent
RAG and TdT expressed
5 and VpreB expression (for Pre-BCR)
Stages of B cell development: (4) Large pre B cell
VDJ Rearranged
L chain genes – Germline
Surface Ig – pre-B receptor chain
Pre-BCR: Following Heavy chain VDJ rearrangement the Pre-BCR is
expressed in Large Pre-B cells by association with a “surrogate
Light chain”
Surrogate light
chain is made
up of gamma5 and
VpreB gene
products.
“The pre-B cell receptor: selector of fitting Ig heavy chains for the B cell repertoire”
Fritz Melchers Nature Rev Immunol 2005
No RAG – prevent further VDJ recomb.
5 and VpreB expressed - PreBCR
TdT still expressed
if recombination events make a functional heavy chain, pair up with surrogate light chain to allow it to be expressed on the cell surface. So now pre b cell receptor expression. Rag turned off to prevent recombination as heavy chain formed. I
