Ch 12: B Cell Activation // Differentiation and Memory Flashcards
Clonal Selection Hypothesis
Each B cell bears a unique BCR, and once stimulated, those B cells will create a wave of clones bearing the same BCR as the original.
The replication of the specific BCRs for specific antigens is a huge power move for adaptive immunity!
Name the three signals of activation for B cells.
- B cell binds to antigen via BCR
- Antigen is internalized, processed, and expressed on MHCII; bind to T cells that gives B cell co-stimulation
- Activating cytokines released by T cells at T-cell/B-cell interface
True or False:
All B cells require T cell help to activate.
False
Once a BCR finds its antigen and undergoes clonal expansion, what cells will the majority of the daughter cells become?
Effector cells; actively fight antigen at the time
What cells are left over after effector cells have undergone clonal expansion from B cell activation?
Memory B cells
How are T and B cells similar in their activation process?
Cell is activated, cell undergoes clonal expansion, some effector cells are formed to attack threat at the time, and some stay behind to form memory cells
What co-receptors are needed to activate B cells in the 2nd signal of activation?
CD40 w/ CD40 ligand
What is the only difference in the three signal activation process between T and B cells?
B cells require cytokines while T cells only need them to differentiate
Just as review, what are the three signals of activation?
- Antigen binding
- Co-stimulation
- Cytokines
What are the two different forms of B cell activation?
- T-dependent responses
2. T-independent responses
What’s the difference between T-dependent responses and T-independent responses?
T-dependent REQUIRES help from T cells to activate
(B-2)
T-dependent do not need T cells (B-1 and MZ)
T-dependent responses by B-2 cells are generated upon recognition by what protein?
TD Antigen
Can you briefly list the three signals of activation for B cells?
- Antigen binds to BCRs
- Exogenous pathway brings antigen on MHCII to bind with TCR; CD40 also binds with CD40L on T cell
- T cell releases cytokines to complete B cell activation
How are T-independent responses generated?
Via exposure to multivalent/polymerized antigens
TI-1 antigens of B1-cell or MZ B-cell bind to B-cells through…
PRRs and mIgs; ANTIGEN doubles as a PAMP
ex: TLRs of B cell may bind w/ PAMP of a bacteria AS WELL AS cross-linking of mIgs with a bacteria
Results in a strong enough response without a T helper cell
TI-2 antigens of B1-cell or MZ B-cell bind to B-cells through…
Cross-linking large numbers of BCRs; usually bound to C3d
Common with repetitive proteins to illicit such strong response on all cross-linked BCRs
ex: Bacterial flagella laying across many cross-linked BCRs may be strong enough to activate w/o T cell
What are iccosomes?
Follicular dendritic cell DENDRITES that are studded with “beads” of antigen/antibody complexes
They are kept on the surface of FDCs to sample the antigens
Do iccosomes appear more helpful in activation of naïve cells OR in CSR and SHM activities that allow the cell differentiation?
More helpful in SHM and CSR bc they aid in LATER differentiation of B cells
B-cell “Sparta”
Where does T-dependent B cell activation take place?
In B cell zone of SLO’s, in specialized regions of lymph nodes or spleen
When B cells are antigen-activated, where do some of them migrate to in SLOs FIRST? What do they differentiate into?
to regions at the border of T-cell and B-cell areas
Its in these areas B cells differentiate into primary foci
Primary foci
Near circulatory branches of lymph node
B cells differentiate quickly from naïve to plasma cells to quickly start making antibody
We can quickly produce Abs in primary foci that leave in the circulatory system. Are they effective, though?
The quickly made Abs are not super specific; they are IgM with moderate binding (4-5 days after exposure)
BUT some is better than none; these Abs will be made better by the immune response
Some B cells do not enter the primary foci to quickly make Abs. Where, then, do they go?
To germinal centers to undergo further differentiation
What 2 processes do B cells undergo when they enter germinal centers of follicules?
Somatic hyper mutation (SHM)
Class switch recombination (CSR)
Somatic hyper mutation (SHM)
Mutation of variable region genes FOLLOWED by antigen selection
Class Switch Recombination (CSR)
Changing antibody classes (from IgM to IgG, etc) via changing out constant regions
Mediated by T helper cells
True or False:
The strongest binding antigen/antibody complex of BCRs is the B cell that wins and proliferates.
True; achieved via hypermutation (SHM)
True or False:
B cells differentiating in primary foci hypermutate and class switch.
False; they only moderately bind to antibody and only secrete IgM
Where do we find naïve B cells in a lymph node?
Traveling from blood vessels to B cell zones aka follicles
Where do we find the primary loci in a lymph node?
Regions at the border for T-cell and B-cell areas
Where do we find germinal centers in a lymph node?
B-cell zone aka follicles
Where do we find T cells in a lymph node?
T-cell zone/Paracortex
Where do we find plasma cells in a lymph node?
released from Primary foci and germinal centers
CXCL13 function
Chemokine produced by follicular stromal cells that draw B cells to the follicle
CXCR5 function
Receptor used by B cells to follow CXCL13 chemokine that help migrate them to lymphoid follicles
BAFF function
Inhibits apoptosis to allow for B cell proliferation (to stay “alive” long enough)
What happens if B cells can’t bind enough BAFF?
They die by apoptosis bc they can’t proliferate following antigen binding
Describe how antigens enter lymph nodes and how antigens can make their way to B cells based on their size.
- Antigens enter lymph node via afferent lymph
- From afferent lymph to subcapsular sinus region
- Antigens that are larger are carried into the SLO to meet B cells via SCSMs, which line the sinus itself
- Smaller antigens diffuse across lymphatic endothelial cells into SLO to interact with B cells
- Some small antigens that diffuse into SLO can also be carried by follicular conduits, which are surrounded by FRCs
- Follicular conduits may have FDCs attached as well as B cells present. The leaky-quality of the conduits allow APCs to take in and present antigen to T cells OR allow B cells to be activated by the traveling antigen
What is the role of FDCs in B cell activation?
They serve as antigen concentration sites; they SAMPLE antigens as they leak from the conduit network
What is the significance of FDCs being antigen concentration site?
They are perfect for activating an adaptive immune response
They can simultaneously trigger a B cell with WHOLE antigen and T cell with processed antigen
What is membrane spreading? How is it advantageous for B cell activation?
When a B cell expands its surface over whatever it is being activated by; where the antigen is coming from
Allows B cell to come into contact with many antigens to get a strong enough signal 1
What are three different sources of antigen B cells utilize for membrane spreading?
Follicular conduit system
FDCs
SCSMs
What is happening during the immunological synapse when B cells interact with their cognate antigen?
Oligomerization of Ag-bound IgM mlcs in B cell membrane
When B cell binds to antigen (by membrane spreading or not), it induces conformational change of the Cµ4 domain, causing more IgMs to interact and bind with the antigen, overall increasing the signal 1 for B cell activation via BCR clustering
How is the immunological synapse formed?
When B cell BCRs bind with CELL-BOUND antigen
As BCRs cluster to form the immunological synapse, what do they further induce to get to signal 2?
BCR clustering after binding with antigen triggers the exogenous pathway to internalize antigen and present it on MHC II fot T cell help
What are the costimulatory signals for T cells, again?
CD80 and CD86; signal 2
cSMAc to pSMAC to actin ring is called….
Supramolecular activating cluster (SMAC)
In what ways can a B cell acquire & engulf its cognate antigen by “stealing” it? (immunological synapse)
- Lysosomal fusion that can cleave bonds between antigen and APC via proteolytic enzymes
- Actin-myosin filaments can act to tug BCRs with antigen attached away from APC.
True or False:
B cells are programmed to respond to chemoattractants.
True; B cells are highly motile
List the the B cell receptors and the ligands responsible for B cell trafficking in the lymph node via chemotaxis.
EBI2 w/ 7α,25-dihydroxycholesterol
CXCR5 w/ CXCL13
CCR7 w/ CCL19 and CCL21
How does CXCL13 traffic B cells in the SLO?
CXCL13 binds with CXCR5 of BCR to draw the B cell into the follicle
B cell scan for antigen in follicle at this point
What cells produce CXCL13?
FSCs
How does 7α,25-dihydroxycholesterol traffic B cells in the SLO?
7α,25-dihydroxycholesterol binds with EBI2 and takes B cell to the outer region of the follicle (marginal zone)
**This only happens when B cells binds with antigen!
How does CCR19 and CCR21 traffic B cells in the SLO?
They bring B cells bound with antigen to B cell/T-cell zone boundary
What cells produce CCL19 and CCL21?
Stromal cells in T cell zones
List the B cell trafficking pathway from 0 hrs of exposure to 4-7 day exposure.
- Antigen surveillance
- Antigen capture
- T-cell encounter
- Clonal expansion
- Plasma cell and germinal center development
What occurs in the primary foci?
B cells w/ an Ag were activated by T cell interaction at the B/T cell border and moved back to follicle border, where they quickly differentiate into IgM-secreting PLASMABLASTS
IgM antibody is produced
Where would you expect to find primary foci in a lymph node?
In the medullary cord region of the node
after 5-6 days of exposure (bc B cells have already been activated by T cell)
True or False:
Primary foci are long-lived immune structures
False; they are found 5-6 days after exposure and die 5-10 days afterwards via apoptosis
What transcription factors dictate whether a B cell will enter a primary focus or germinal center?
IRF-4 and BLIMP-1 = primary foci formation
Pax-5 and Bcl-6 = germinal center formation
How many days before primary foci can be observed following antigen exposure?
5-6 days
How many days before there is a peak in primary foci activity?
7-8 days
How long do these plasma cells tend to live before dying by apoptosis?
14 days
What occurs in germinal centers?
B cell/antigen sparta!
B cells that didn’t form into primary foci at B/T cell border region are moved back to the follicle, where SHM and CSR occurs for the best antigen binding possible for B cells.
where would you expect to find germinal centers in a lymph node?
Follicle/ B cell zone
What is occurring in the dark zone of a germinal center?
Intense proliferation of B cells that are densely packed, appearing dark on histological samples
Somatic hypermutation is taking place; proper BCRs are being made to bind the best with antigen
What is occurring in the light zone of a germinal center?
Here B cells are trying to grab as many antigens as they can via help of FDCs interspersed in this area
Here, B cell affinity for antigen has greatly improved, and the B cells that failed to bind better died. Lighter area bc its only for the survivors.
How does the mutation rate of Ig variable gene segments compare to the normal background mutation rate of cells?
B cells are experiencing extremely high rates of mutagenesis (1 mutation per thousand bp per generation)
100,000 x more than the usual mutation rate, which is 1 mutation per HUNDRED MILLION bp per generation
Summarize what SHM is in one sentence.
Somatic hypermutation occurs in the germinal center where BCRs are going through intense mutation of their variable regions to strongly bind to antigens in order for better B cell survival.
Summarize what CSR is in one sentence.
Class switch recombination is where the constant region of the BCR changes from the µ constant region to other segments, resulting in new antibody isotypes being secreted other than IgM.
What enzyme leads to SHM?
Antigen-induced cytidine deaminase (AID)
What does AID do overall?
Holistically, AID produces the opportunity for mutations to occur; the deaminase aspect of the enzyme turns cytidine into uridine, causing many mutations
The mutations arise as a response to this bp change by DNA replication machinery
How does AID lead to SHM?
- The converted C is now U, so DNA polymerase reads this as T, so A is paired with it (point mutation)
- Short-patch base excision; polymerase excises U but any nucleotide could replace it, causing point mutations
- Mismatch repair; a chunk of bp’s are taken out where the U was (patch of mutations!)
How does the AID enzyme know to act on certain areas of the genome?
AID can only act on Ig gene segments
Mutational hot spots
A specific sequence motif
DGYW/WRCH
-G–/–C- G and C HAVE to be in this motif for AID to work
How do B cells gain higher affinity for antigen in the germinal center over time?
B cells are going through intense hypermutation for their BCRs to bind antigen the strongest. Those bindings are selected for to see who’s BCR has the best Ag affinity. The better affinity a BCR has for Ag, the more survival signals it gets from cytokines of T helper cells
True or False:
High affinity B cells can steal antigen from lower-affinity B cells.
True; actomyosin filaments of the B cell can pull antigen away if BCR/Ag binding is stronger
SPARTA
What happens to autoreactive B cells in the SLO?
Autoreactive BCRs get so overwhelmed by our own peptides that all the BCRs of B cells in the SLO don’t have any surface BCRs available.
Therefore, B cell loses BCR signaling, exits the cycle, and dies via apoptosis (die by neglect)
What happens to autoreactive B cells in the periphery?
Autoreactive B cells can’t achieve T cell costimulation; there will be no T cell help, so they die = ANERGY
How does AID lead to CSR?
There are a ton of tandem repeats of G-rich sequences, therefore a bunch of chances for AID to change Cs to Us, leading to double stranded breaks. which is where the constant region switches will occur
B cells receive costimulatory signals from ______ on T cells to engage in CSR.
CD40L
What is the link between cytokine signaling, switch regions, transcription of sterile RNAs, and double-stranded breaks for BCR constant region mutations?
ask Dr. Rhodes
What cytokines are critical for maintaining long lived plasma cells in bone marrow?
APRIL (survival signals) and CXCL12 (chemokine; keeps plasma cell in bone marrow)
What is APRIL?
A proliferation-inducing ligand, produced by eosinophils and megakaryocytes
What is the average half-life of a plasma cell OUTSIDE of the bone marrow?
4.5 months; shorter lifespan
How are memory B cells different than naïve B cells?
- Memory cells respond quicker and stronger when cognate antigen is found; they’ve seen it before
- Memory B cells live longer, naïve B cells are short-lived bc they only rely on Ag, and when it’s gone, they die by apoptosis
- Naïve B cell has IgM, Memory has IgG
- Memory has high Ag affinity (went through SHM and CSR)
How could you tell if a patient had been recently exposed to a pathogen (a week or less) or if they had been exposed three weeks ago using only an antibody test?
If patient was recently exposed, there would be more IgM antibodies in early primary response
If patient has been exposed three weeks ago, we’d see more IgG predominantly
True or False:
All antibody-producing responses need T cell help.
False; bc not all B cells need help activating by T cells, such as B1 cells or MZ B cells
What experiment showed that antibodies could be produced even when no T cells are present to provide activation signals?
Freddy! Nude mice that have athymia (no thymus/no T cells) were found to still be able to respond to T-independent antigens with antibodies
Although Abs can still be produced w/o T cell, they Abs themselves are low affinity IgM, still eliciting a low immune response (lack of SHM and CSR)
What antigens are responsible for B cell subset activation w/o T cells help?
Polyvalent, repeating structures
PAMPS of microbes or blood borne pathogens
What types of B cells are known for recognizing T-independent antigen?
B1 B cells and MZ B cells
What type of antigens are considered TI-1 antigens?
Typically bacterial cell wall components (PAMPS)
activate BOTH BCR and PRR on B cell
What is a clinical consequence to having too much TI-1 Ag binding to PRRs of all B cells?
Septic shock; TI-1 Ag can be mitogenic to all B cells at high doses (gram neg. bacteria)
What occurs when someone is exposed to low amounts of TI-1 antigen?
The PRRs of B1 or MZ B cells cannot bind enough antigen to activate them
How can we still get T-independent B cell activating if TI-1 Ag is in low dosage?
B cells also have Ig receptors that can bind to the antigen and cross link it to the PRRs, making up for what couldn’t bind to the PRRs.
Why don’t areas of our body constantly in contact with gram neg, bacteria (like our gut) reslut in over-stimulated immune responses?
Mucus layer and T reg cells
What type of antigens are considered TI-2 antigens?
Polymeric protein Ag and capsular polysaccharides
Bc these antigens are larger, they can cross link many mIgM BCRs across the B cell surface as one identical antigen for one effective response
Compare & contrast the three types of antigens listed on slide 55.
TD= protein based, give us different Ab (not polyclonal=doesn’t activate several types of B cells)
TI-1= reacted to by B1 cells
TI-2= reacted to by B1 and MZ B cells
- B1 B cells and MZ B cells are more innate
Explain how CD22 & SHP-1 work to negatively regulate B cell activation.
When B cells are activated, CD22 is immediately phosphorylated and binds to SHP-1 tyrosine phosphatase. SHP-1 affiliates with the cytoplasmic tail of CD22, which then dephosphorylates BCR signaling (ITAMS)
True or False:
The drop in antigen will allow SHP-1 to work faster and more efficient to halt B cell activation.
True; ITAM from BCR drops, ITIM helps stop activation
Under what conditions does CD22 & tyrosine phosphate “win” when it comes to inhibiting BCR signaling?
Only when antigen levels decline (at the end of the immune response)
