B Cell Biology (Affinity Maturation, Memory, Plasma Cell Homeostasis) Flashcards
Why are memory cells able to respond quickly?
proteins necessary for division (Cdks etc) are already synthesised
cells preloaded with effector molecules eg IFN-γ, TNF-α
elevated levels of signalling compounds and transcription factors (eg Lck in T cells)
higher frequency than naive cells (a 1000 fold increase)
higher Ab affinity (affinity maturation)
some are resident in tissues
epigenetic priming: effector genes are much more accessible eg e.g., histone acetylation, chromatin remodeling
Memory cells express higher levels of cytokine receptors, such as IL-7R and IL-15R
- makes them more sensitive to environment
What are some characteristics of memory B cells?
- express switched isotypes (some remain IgM+)
- express somatically mutated Ig V genes
- express higher affinity Ig
- lower activation thresholds
- increased levels of adhesion molecules (CD44, LFA-1) = improved interactions w APCs and increased recruitment to site of inflammation
plasma cells are not memory cells as they have finite lifespans
Why do memory T cells change the isoform of CD45 they express?
- naive resting T cells express high molecular weight forms of CD45 (RA, RB, and RC)
- lose expression of these after antigenic stimulation
– arise in differences with alternative splicing
– shorter isoform brings the phosphatase domain of CD45 closer to the TCR signaling complex
– Enhances the dephosphorylation of inhibitory sites on Lck , amplifying downstream signaling
= more efficient and rapid T cell activation in memory cells
– used as a marker for memory T cells, however reversion to RA, RB, or RC expression does occur
How does memory cell differentiation occur?
asymmetry occurs at a population level rather than a cellular level
ie populations exposed to different microenvironments diverge
low Ag +/- cytokines = memory
high Ag + cytokines = effector
What TFs are important for differentiation of CD8 into SLEC or memory cells?
Blimp1: induces activated T cell/early effector to become short lived effector cell (SLEC)
– expresses low IL-7R, important survival factor
Bcl6: induces activated T cell/early effector to become a memory precursor effector cell, continued exposure = memory cells
– actively downregulates Blipm1
What are the different types of B cell and what induces them?
short-lived plasma cells: Blimp1
germinal centre B cells: exposure of activated B cells to Bcl6
– these B cell produce Bcl6 for upkeep of germinal centre
long lived plasma cells: exposure of Blimp1 to gc B cells
memory B cells: arise from gc B cells
What are two populations of memory cells?
- those that differentiate and respond upond reinfection – effector memory
- those that renew memory pool
if all memory cell differentiated upon reinfection then the memory would be lost
T cell memory is impaired in mice without B cells.
experiments show that compared to WT mice, mice without B cells have a lower number of memory T cells after 12 weeks
How do B cells act as decoys to create memory T cells?
During T cell contraction, T cells express both Fas and FasL on their surface, clearing cells as they can cause each other to apoptose
B cells also express Fas, so can act as decoys to cause T cells to become memory in the germinal centre due to interactions w CD4
some B cells may be lost
this is hypothetical
How are memory B cells tracked in vivo?
phycoerythrin bins B220 memory B cells
How are memory T cells tracked in vivo?
MHC class II tetramers can bind to the T cell receptors (TCRs) of CD4+ T cells that specifically recognize the peptide-MHC complex
What is homeostatic regulation of lymphocytes?
all lymphocytes are under constant selective pressure as the size of the lymphoid system is controlled within strict limits
– if many lymphocytes are injected , the immune system does not incorporate them all
– after a couple of days it returns to its normal size
– there appear to be a finite number or niches in the immune system that lymphocytes can occupy
eg access to DCs and cytokines
once all niches are occupied, older memory cells are deleted
How do we know about lymphocytic survival niches?
when memory T cells (from TCR transgenic mice) are transferred into immunodeficient hosts (eg RAG -/- or SCID) the number of niches is not longer limiting
lifespans may be extended compared to that in a complete immune system
What are some factors influencing memory survival?
- antigen persistence (on FDC or other sites eg persistent viruses)
- re-exposure to priming agent (pathogen)
- exposure to a cross-reacting organism/antigen
- non-specific signals (cytokines)
oldest memory cells lose survival receptors eg IL-15R (homeostatic attrition)
Why does memory not equal protective immunity?
memory is an accelerated, enhance response
– may not give absolute protection from re-infection
protective immunity provides complete protection from re-infection
– provided by effector cells and tissue-resident effector memory cells
– dependent on antigen
How is measles an example of memory?
immunity to measles was long lived
- re-exposure to virus was not essential for long term protective immunity
not evidence for antigen free survival
- persists within the body (CNS)
How does the BCR signal?
BCR = membrane Immunoglobulin (mIg)
signalling heterodimer composed of Igalpha and Igbeta subunits (CD79a and CD70b)
Both have ITAMs in their cytoplasmic tails
When an antigen binds to the BCR:
receptor cross-linking occurs, bringing multiple BCRs together on the membran
This clustering brings Igα/Igβ molecules into close proximity, facilitating their phosphorylation by
Src-family kinases (e.g., Lyn, Fyn, or Blk) which phosphorylate the ITAMs
Phosphorylated ITAMs act as docking sites for downstream signaling molecules eg Syk
What is the structure of the B cell surrogate light chain?
composed of two non-polymorphic proteins:
- VpreB: mimics variable region of Ig light chain
- λ5: mimics constant region of Ig light chain
these two proteins pair w a successfully rearranged Ig heavy chain (igH) to form the pre-BCR
What is the role of the surrogate light chain in B cell development?
– allows the testing of a newly rearranged heavy chain for functionality before the light chain genes undergo rearrangement
– pre-BCR signals the pre-B cell to proliferate and halt further heavy chain rearrangement, a process known as allelic exclusion
– After successful heavy chain testing, the surrogate light chain signals for the rearrangement of light chain genes (κ or λ), ultimately leading to the formation of a complete BCR
– If the heavy chain fails to pair with the surrogate light chain, the cell undergoes apoptosis, ensuring that only B cells with functional pre-BCRs progress in development
What are transitional B cells?
immature B cells that have recently exited the bone marrow and entered the peripheral blood and secondary lymphoid organs
T1:
– short lived and highly sensitive to apoptosis
– low CD21 and CD23 expression
which mature into
T2
– more resistant to apoptosis
– increased expression of CD21 and CD23
What is BAFF?
B cell activating factor of the TNF family
critical survival factor for the B cell lineage
blocking BAFF causes loss of mature B cells from secondary lymphoid organs
What are the different areas of the spleen?
PALS
- T zone
primary follicle
- B cells
marginal zone
- IgM, IgD, and double +ve B cells
germinal centre (within follicle)
- mature B cells
What are B1 B cells?
- evolutionarily early
- make cross-reactive antibodies
- make T-independent antibody responses
- make mainly IgM and IgG3
What are B2 B cells?
- majority of B cells
- make T dependent Ab responses
- make IgM and all switched isotypes
What are the 3 modes of B cell activation?
T-independent:
TI-1: mitogen (LPS, bacterial products)
TI-2: Ig cross-linking (bacterial polysaccharides)
T-dependent:
TD: protein antigens
A monovalent Ag does not cross-link the BCR, how does it induce signalling?
Dissociation Activation Model
BCRs are oligomeric clusters in their resting configuration
– In this state, the receptors are associated in inactive complexes, and Lyn, a key kinase for signalling, remains inactive
A monovalent antigen binds to a single BCR, disrupting the oligomeric complexes and “opening” the BCR
This disruption allows Lyn to associate with the BCR, leading to the initiation of downstream signalling
Thus, a signal is generated even with monovalent antigens
however crosslinking model is primary mode of activation
How does complement help B cells activate?
If antigen is tagged w complement, eg C3d, it reduces amount of antigen needed for B cell activation by up to 1000-fold
Why is clustering of receptors crucial for BCR signalling?
clustering of Ag receptors allows receptor-associated kinases to phosphorylate ITAMs
Syk binds to doubly phosphorylated ITAMs and is activated on binding
What does binding of antigen do in the context of antigen presentation?
up-regulation of MHC class II and co-stimulatory molecules (B7 = CD80/86) and facilitates uptake/processing of antigen
How is T-I activation of B cellls made into a strong signal?
help from T cells
- antigen activation
- co-stimulation
– CD80/86 binding to CD28 on T cells
– CD40 binding to CD40L
without this, B cells would undergo anergy or death/deletion (i.e tolerance)
What is the difference between B and T cell antigen recognition?
B cells can recognise free antigen, which has differently exposed epitopes due to protein conformation
T cell recognises short, linear epitopes bound to MHC
What is linked recognition?
A B cell recognises a particular epitope on an antigen (eg A), but can present multiple epitopes from that same antigen (eg E)
An anti-E T cell that doesn’t recognise A can still recognise E that is presented by the anti-A B cell
How has linked recognition been shown experimentally
hapten, a small molecule that does not elicit an immune response on its own, but ,must be linked to a larger carrier protein (Y)
anti-hapten B cell
anti-Y T cell
B cell recognise hapten which is linked to carrier protein Y, which elicits a T cell response
if carrier protein X is used, no T cell activation
== linked recognition
What are different outcomes of B-T cell interaction?
- Ab response
(these occur in germinal centres)
- proliferation
- Ig class switching
- somatic hypermutation
- antibody production
- formation of memory cells
How are helper T cells activated?
interactions with dendritic cells
T cell CD40L -> DC CD40
Give examples of cytokines which direct Ig isotype switching?
IFNgamma –> IgG2a or IgG3
TGF-beta –> IgA or IgG2b
IL-4 –> IgE or IgG1
IL-2, 4, 5 –> IgM
works by regulating the transcription of switch regions (S regions) upstream of the C genes
This makes specific S regions accessible to AID, determining which antibody isotype the B cell will produce
What are the different outcomes for immature B cell of binding Ag in BM?
recognition of multivalent self molecule
– arrest of development and continued light chain rearrangement
– if new receptor is still self-reactive, B cell undergoes apoptosis
– otherwise immature B cell migrates to periphery
recognition of soluble self molecule
– migration of B cell to periphery
– put into an anergic state
low affinity non-cross-linking self molecule
– migrates to periphery
– B cell matures as normal, “clonally ignorant”
– because interaction w self-antigen is weak and does not respond to them under normal physiological conditions
– this prevents unnecessary elimination of B cells that could recognise antigens structurally similar to self antigens
How do B and T cells move to their respective areas in the lymph node from the blood?
localisation controlled by differential expression of chemokine receptors responding to opposing chemokine gradients
eg T zone
- SLC/CCL21
– SLC = SLT chemokine
- ELC/CCL19
– ELC = EBV induce molecule 1 ligand chemokine
eg follicle
- BLC/CXCL13
– BLC = b lymphocyte chemokine
What are FDCs?
follicular dendritic cells
derived from perivascular precursors, not haematopoietic origin
present antigen in germinal centres
What are properties of FDC antigen?
- Antigen can be stored in native form for very long periods (>1 year).
- FDC do not internalise antigen or present it to T cells.
- Intermediary APC required for presentation to T cells
How do antigens enter lymph nodes to be given to FDCs and presented to B cells?
- Antigens (eg. bacteria, immune complexes) enter lymph nodes in the subcapsular sinus (from afferent lymphatics that drain tissues)
- Antigens captured by macrophages in the subcapular sinus
- B cells acquire antigen from these macrophages and transport it to FDC. Initially, they use CR2 to pick up antigen (some Ag-specific cells use BCR)
- antigen stored in beads: iccosomes
- Antigen-specific B cells tear antigen from the FDC (some FDC membrane is also acquired)
What are Tfh?
T follicular helper cells
- needed for GC response
- dependent on B cells
express Bcl6, important for generation of GC
– incorporate into GCs
differentiate from CD4 T cells
What is the function of germinal centres?
- clonal expansion of Ag-specific B cells
- affinity maturation
– somatic hypermutation
– antigenic selection
crucial components of memory B cell formation and long-lived plasma cell populations (in BM)
What two populations of B cells present in GCs?
centroblasts
– in cell cycle
– localised in dark zone
– proliferation/hypermutation
centrocytes
– out of cell cycle
– localised in light zone
– selection -> rescue
What are the two stages of selection in a GC?
Stage 1
– antigenic selection on FDC
– bcl-2 independent short term rescue
Stage 2
– induction of CD40L signal
–> bcl-dependent longterm rescue
– signal and exit to the memory pool
What evidence shows FDC are crucial for mutation and selection of B cells?
LTalpha KO mice have no FCD
– exhibit mutation and selection but much less efficient
selection mediated by FDC bound antigen
Compare a mature B cell and a plasma cell.
Mature B Cell
Primary Function: Antigen recognition and presentation.
Antibody Expression: Membrane-bound (as part of the BCR).
Morphology: Small, with a dense nucleus
Location: Secondary lymphoid organs (e.g., lymph nodes, spleen) and circulating in the blood.
Lifespan: Variable, ranging from weeks to years depending on activation and differentiation status.
Plasma Cell
Primary Function: Antibody secretion
Antibody Expression: Secreted antibodies
Morphology: Large cell, abundant cytoplasm
Location: Bone marrow, mucosal tissues, or secondary lymphoid organs (for short-lived plasma cells).
Lifespan: Short-lived (days) in secondary lymphoid organs or long-lived (years) in the bone marrow
undergone class switching and somatic hypermutation in GC
