Handout 12 part 2: B cell Activation and Antibody Production Flashcards
How is B cell activated initiated?
by specific recognition of Ags by BCRs
How is B cell proliferation and differentiation stimulated?
Ags and other stimuli stimulate proliferation and differentiation of specific B cell clone
What are the various types of cells that B cells can differentiate into after they have proliferated?
Can differentiate into plasma cells that produce IgM other Ig istoype may undergo affinity maturation persist as memory cells
Summary of humoral immune response (how B cells proliferate and differentiate)
Ag recognition by Naive IgM+, IgD+ B cell
Activation of B cell by Helper T cells and other stimuli
Proliferation:
Plasma cell (IgM)–>Ab secretion
IgG expressing B cell–>isotype switching
High affinity Ig expressing B cell–>Affinity maturation OR Memory B cell
What is the result of alternative processing of a primary RNA transcript?
formation of mRNA for the membrane OR secreted form of the mu heavy chain
What does B cell differentiation result in?
results in increasing fraction of the mu protein produced as the secreted form
What parts of the IgM do the following terms refer to:
TP
TM
CY
TP=tail piece
TM=transmembrane
CY=cytoplasmic segments
What are Cmu1, Cmu2,Cmu3, and Cmu4?
They are four exons of the Cmu gene
Define the following acronyms: CDR Fab Fc VH VL CH/L
CDR=complementarity-determining region Fab=antigen binding fragment Fc=crystallizable fragment VH=variable domain of heavy chains VL= variable domain of light chains CH/L=constant domain of heavy/light chains
Where is the CDR found in the Ig? Where does the Ag bind? Which region is associated with effector functions? Which end of the Ig has the amino end and which has the carboxyl end?
CDR found in variable regions of heavy and light chains
Ag binds to Fab region of Ig
Fc portion is associated with effector functions
Fab (Ag binding region) has amino end, and Fc fortion of heavy chain has carboxyl end
To which domain does C1q bind? Which domains are found in all Igs? Which domains are specific to only a few Igs? What are these Igs?
C1q binds to CH2 domain
CH1, CH2 and CH3 are found in all Igs but CH4 is ONLY found in IgE and IgM!
In what kind of immune response (T-dependent or T-independent?) do B cells undergo heavy chain isotype class switching? What are the Igs that undergo class switching and what kind of heavy chains do these Abs produce?
Activated B cells that express IgM and IgD in T-cell DEPENDENT responses undergo heavy chain isotype (class) switching
They produce Abs with heavy chains of classes like gamma, alpha and epsilon
Where does isotype switching primarily occur? What kind of cells drives this process?
isotype switching mainly occurs in germinal centers and are driven by follicular T helper cells
some isotype switching may also occur in B cells in extrafollicular foci, driven by extrafollicular Th cells
What is the advantage of being able to switch Ab isotypes?
B cells producing different Ab isotypes provides plasticity in humoral immune responses
Abs of different classes perform different effector functions and are involved in defense against different types of pathogens
Which part of the Ig is involved in class-switching?
Class-switching occurs in response to changing the constant regions of the heavy chains
How is specificity of class-switched Abs maintained?
Specificity is determined by the variable regions which remains unaltered during class-switching
B cells activated what kind of signals undergo Ig isotype switching?
B cells activated by Th cell signals–CD40L, cytokines
Each class of Abs mediates distinct effector functions. What are the principal effector functions of each of the following classes of Abs: IgM IgG (subclasses: IgG1, IgG3) IgE, IgG4 IgA
IgM: Complement activation
IgG subclasses (IgG1, IgG3): Opsonization and phagocytosis; complement activation; neonatal immunity (placental transfer)
IgA: Mucosal immunity (transport of IgA through epithelia)
What cytokine is needed to convert an activated B cell into each of the following classes of Abs:
IgG subclasses (IgG1, IgG3)
IgE and IgG4
IgA
IFN-gamma needed to switch into IgG subclasses (IgG1, IgG3)
IL-4 needed to switch into IgE and IgG4
TGF-beta, APRIL, BAFF in mucosal tissues needed to switch into IgA
How is isotype switching regulated in response to different types of microbes?
Regulated by cytokines produced by Th cells that are activated by microbes
IFN-gamma induces B cell switching to ___ and IL-4 induces switching to ___
IgG (Th1)
IgE (Th2)
What is the function of IgG Abs?
They respond to viruses and intracellular bacteria and involves IgG Abs which block entry of microbes into host cells and promote phagocytosis by macrophages
Intracellular pathogens activate which subset of helper T cells? What cytokine does this subset of T cells produce?
Activate Th1 cells which produce IFN-gamma and also induce IFN-gamma follicular T helper cells to make more IFN-gamma
What is the major function of IgE?
Response to helminths
Participates in eosinophil and mast cell-mediated elimination of the helminths
Helminths influence Tfh cell differentiation into ____ cells which produce ___ cytokines in the germinal center reaction
IL-4+ Tfh cells
Th2-type cytokines
What is the role of anatomical location in isotype switching?
B cells in different anatomic sites switch to different istoytpes which is controlled by distinct cytokines produced at each site
In which anatomical site do B cells switch to IgA? Why is this site favored for IgA over others?
B cells in MUCOSAL tissue switch to IgA because it is the Ab that is most efficiently transported through epithelia into mucosal secretions. Here it can defend against microbes that try to enter through the epithelia
What cytokines regulate the switching of B cells into the IgA subtype? What cells produce each of these cytokines?
TGF-beta produced by Th cells, T reg cells and other cell types
BAFF of the TNF family
What is the role of CD40-CD40L signaling in isotype switching?
They work with cytokines to induce switching
CD40 engagement induces enzyme activation-induced deaminase (AID) which is crucial for isotype switching and affinity maturation
What happens when there are immunodeficiencies of CD40-CD40L?
Studies of mice and humans lacking CD40 signaling and AID show that it is needed for isotype switching
Lack of CD40-CD40L axis results in response mostly by IgM Abs to protein Ags and limited class switching to other isotypes
Before isotype switching, what is the first Ig that the mature B cells produce? Where are the rearranged VDJ seqences positioned and how rearrangement controlled?
Mature B cell first produces IgM immunoglobulins
Rearranged VDJ sequences are positioned directly adjacent to other C genes
Every C gene is preceded by a switching (S) sequence that controls the rearrangement process
What is CSR (Class-Switch Recombination)?
CSR is a switch of the Ig isotype from IgM/IgD to IgG, IgA or IgE with similar Ag specificity but with different biological properties
On what kinds of cells does CSR take place? What specific changes occur during CSR?
CSR takes place in activated B cells
Changes the CH gene that will be expressed from the C mu region to one of the other CH genes
Where specifically in the activated B cell does CSR take place? What is the composition of this area?
CSR takes place between two switch regions (S) comprised of repetitive sequences of palindrome-rich motifs and results in looped-out deletion of the intervening DNA segments
What occurs before CSR? Why is this process important?
Expression of germline transcripts initiated from intronic promoters and regulated by various cytokines
This opens the chromatin structure of a specific S region and renders it accessible to the putative recombinase
Key enzyme required for isotype switching and affinity maturation is what?
Activation-induced cytosine deaminase (AID)
How is AID expression activated?
by CD40 signals from Tfh cells
What does AID specifically do to induce isotype switching and affinity maturation?
It deaminates cytosines in single stranded DNA templates, converting residues to uracil (U) residues
Then, Uracil N-glycosylase (UNG) removes U residues to generate abasic sites where the APE1 endonuclease creates nicks thatlead to a double-stranded break
Seqeuence of events for CSR (Class-Switch Recombination)
Transcription–>Cytidine deamination–>Base-exision repair or mismatch repair–> synapsis (including H2AX, 53 BP 1, ATM,LRI, MLH1, DNA-PKcs) and end joining (NHEJ)–> Productive CSR
What is affinity maturation?
Process that leads to increased affinity of Abs for a particular Ag
Affinity maturation is observed only in Ab responses to what kind of Ags?
T-dependent protein Ags
What interaction is necessary for somatic mutation to be initiated for affinity maturation to occur?
CD40:CD40L interactions
Where and how does affinity maturation occur?
In germinal center reactions, somatic hypermutation of Ig V genes and selection of B cells with high-affinity Ag receptors result in production of Abs with high affinity for the Ag
Which kind of Abs are efficient at neutralizing and eliminating microbes? (high or low affinity Abs?)
Abs that have increased ability to bind Ag (high affintiy Abs)
Process of affinity maturation going from low to high affinity summary:
Low affinity Ab–>somatic mutations in Ig V genes–>selection of high-affinity B cells–>mutations result in high affinity Abs
Affinity maturation in GC
Ag specific B cell in dark zone of germinal center proliferates and undergoes point mutation in its V region of H/L chain
Each proliferated B cell interacts with Ag on FDC in the light zone
Only the mutated Ig with higher affinity binds Ag presenting FDC (amino acid switch leads to a 10 fold increase in affinity) and survives
the others die via apoptosis
Cycle repeats–the Ig goes back to the dark zone and process occurs again
In affinity maturation, where do Ig V genes undergo point mutations?
In proliferating GC B cells in the dark zone
Is the rate of mutation of B cells in the dark zone similar to other spontaneous mutations?
No, mutations occuring for the purpose of affinity maturation are much higher than spontaneous mutations; the rate is 1 in 10^3 which is 1000x more that in other genes
This is why this process is called somatic hypermutation
In somatic hypermutation, how many mutations occur per cell division?
one mutation per cell division accumulates in V regions:
VH+VL=700 nucleotides + Mutation rate is 1 in 10^3 base-pairs=1 mutation per cell division
How many nucleotides does the Vh and Vl genes in each B cell contain?
700 nucleotides
Can IgV gene mutations occur in progeny of individual B cells?
Yes, this is how a B cell clone can accumulate more and more mutations during its life cycle in the germinal center.
The nucleotide sequences of IgG Abs derived from one clone of B cells can have as much as __% germline sequence mutated which adds upto ___ amino acid substitutions
5, 10
In what region of the Ig does the mutations occur?
Clustered in the V region in the Ag-binding complementarity-determining regions
Which Ab has more mutations–IgG or IgM? What does this mean in terms of affinity?
IgG; the presence of mutations correlates with increasing affinities for the Abs for the Ag that induced the response
In terms of immunizations, when do mutations in V genes increase?
Mutations in V genes increase with time after immunization and with repeated immunizations
The affinities of the Abs produced also increases with more mutations
Determined by experiments with hybridomas produced from spleen cells of mice immunizes with a hapten–oxazoline coupled with a protein obtained after 2ndary and tertiary immunizations with the same Ag
How are B cells rescued from apoptosis?
Binding of the B cells to Ag displayed on follicular DCs saves B cells from apoptosis
B cells also may present Ag to germinal center Tfh cells which promote B cell survival in a CD-40 dependent manner
The amount of Ag available decreases during an immune response so this means that what kinds of B cells have a selective advantage for survival?
B cells with the highest affinity for Ag; this leads to an increase in the affinity of Abs for Ag as the humoral immune response progresses
B cell selection and survival summary
B cell acitvation by protein Ag and helper T cells–>
Induction of AID and migration into germinal center–>
B cells with somatically mutated Ig V genes and Igs with varying affinities for Ag–>
B cells with high-affinity Ag receptors are best able to recognize Ag on follicular DCs and bind Ag and present it to Tfh cell–>
Only B cells with high affinity Ag receptors are selected to survive
Ag binding to BCR occurs in the ___. B cells that are selected to survive in GCs produce Ig of increasing affinity for the Ag. This selection process results in ____
Germinal center
affinity maturation of the Ab response
What is the site of apoptosis of B cells generated by somatic mutation that do NOT express high affinity receptors?
Germinal centers
Where do the B cells that have undergone somatic mutation migrate into?
They migrate into the FDC-rich light zone of the germinal center
B cells with high affinity receptors for Ags best bind to Ag when it is present at low concentrations by follicular DCs. What are the mechanisms by which certain B cells survive preferentially?
- Ag recognition induces expression of anti-apoptotic proteins of the Bcl-2 family
- High-affinity B cells will preferentially endocytose and present the Ag for Tfh cells in the GC which signal via CD40L to promote survival of B cells
- Some Tfh cells express FasL which can recognize Fas on B cells and induce apoptosis. High affinity B cells may activate endogenous inhibitors of Fas when their BCRs recognize Ag and are protected from apoptosis. Low affinity B cells are killed
Many tumors of B cells, B cell lymphomas develop from __ __ _ ___
germinal center B cells
What are B cell lymphomas and tumors caused by?
chromosomal traslocations of oncogenes into Ig gene loci
DNA breaks associated with somatic hypermutation and isotype switching facilitate incorporation of oncogenes
How does autoimmunity occur in germinal centers?
Germinal centers contribute to autoimmunity
Somatic mucation may produce self-reactive B cell clone in germinal center
What are plasma cells? When are they generated (what signals are necessary for its production?)
Plasma cells are terminally differentiated B cells committed to abundant Ab production
They are generated after activation of B cells through signals from BCR, CD40, TLRs, and other receptors including cytokine receptors
What are the two types of plasma cells? What are the differences between the two types of plasma cells?
Short-lived plasma cells:
generated in T-INDEPENDENT responses or in EARLY T-dependent responses in extrafollicular B cell foci
found in secondary lymphoid organs and peripheral non-lymphoid tissues
Long-lived plasma cells:
generated in T-DEPENDENT GC responses to protein Ag
generated by signals from the BCR and IL-21 via a stage of their precursors called plasmablasts
Where do long-lived plasma cells differentiate?
Plasmablasts generated in germinal centers enter the circulation and home to the bone marrow where they differentiate into long-lived plasma cells
2-3 weeks after immunization the __ ___ becomes a major site of Ab production
bone marrow
Plasma cells are maintained by ____allowing cells to survive for long periods, often as long as the life span of the host
BAFF
How long do plasma cells secrete Abs? Does the Ag need to be present to produce Abs?
Plasma cells secrete Abs for months or even years even after the Ag is no longer present so that the Abs can provide immediate protection if the Ag is encountered later
It is estimated that almost __% of Ab in the blood of a healthy adult is produced by long lived plasma cells and is specific for Ags that were encountered in the past
50
Where are memory cells generated? Memory cells are generated in response to what kind of Ags?
Germinal centers
In response to T-dependent protein Ags
They can survive for long periods without continuing Ag stimulation
Memory B cells express high levels of anti-apoptotic protein. What is the name of this protein?
Bcl-2
Memory cells typically express _______ Ag receptors and Ig molecules of ___ isotypes
high-affinity (mutated)
switched
Abs production is greatly accelerated after ____ exposure to Ags and this can be attributed to activation of ___ cells in ___ ____
second, memory, germinal centers
Where are memory cells found?
Some memory cells remain in the lymphoid organ where they were generated whereas others exit germinal centers and recirculate between the blood and lymphoid organs
Effective vaccines against microbes and microbial toxins must induce 2 important processes. What are they? When does this occur
Affinity maturation
Memory B cell formation
Occurs ONLY if the vaccines are able to activate helper T cells
Concept of conjugate vaccines has been applied to the design of vaccines for _____ ______ which is incapable of stimulating T cells
capsular polysaccharides
In capsular polysaccharide vaccines what binding/linkage occurs and what is this complex called?
polysaccharide is covalently linked to a foreign protein to form the equivalent of a hapten-carrier conjugate
These are called conjugate vaccines and more readily induce high affinity Abs and memory cells
What are thymus independent Ags/responses?
non-protein Ags such as polysaccharides and lipids that stimulate Ab production in the absence of Th cells
Abs that are produced in the absence of Th cells–are they usually of high affinity or low affinity? What Ig do these Abs consist of?
They are of low affinity
Consist mainly of IgM with limited isotype switching to some IgG subtypes and also to IgA
Thymus-dependent Ag vs. Thymus independent Ag Chemical nature: Isotype switching: Affinity maturation: Secondary response (memory B cells):
Chemical nature: T dependent are proteins, T independent are polymeric Ags–especially polysaccharides, glycolipids and nucleic acids
Isotype switching: T dep. shows lot of isotype switching (IgM, IgG, IgE and IgA)
T ind. shpws low level of switching
Affinity maturation: T dep=yes; T ind=little or No Secondary response (memory B cells): T dep=yes, T ind=only seen with some polysaccharide Ags
The differences between T dependent and T independent Ags largely reflect the influence of ___ cells in responses to protein Ags
Th cells
Most TI (T independent) Ags are multivalent. What does this mean? What is the benefit of being multivalent?
Means that the Ags are composed of repeated identical Ag epitopes/subunits
Multivalent Ags allows for maximal cross-linking of the BCR on specific B cells, leading to activation without a requirement for cognate T cell help
Where are TI Ab responses initiated?
spleen
bone marrow
peritoneal cavity
mucosal sites
What kind of B cells are involved in Ab responses to TI Ags?
marginal zone and B-1 B cells
What kind/class of Ags do Marginal zone B cells specifically respond to?
Responds to polysaccharides
What kind of cells do marginal zone B cells differentiate into? (Long-lived or short-lived plasma cells?) What class of Ig do these cells produce?
short-lived plasma cells that produce IgM
TI Ags may persist for prolonged periods on the surfaces of ___ __ ___ in the spleen where they are recognized by specific B cells
marginal zone macrophages
B-1 cells also respond to TI Ags. What tissue are these cells primarily active in?
peritoneum
mucosal sites
What are the most important TI Ags?
polysaccharides
glycolipids
nucleic acids
Why are TI Ags not recognized by CD4+ helper T cells?
Because they cannot be processed and presented in association with MHC molecules
Many TI Ags that are polysaccharides can activate the complement system. Which pathway do they typically use for complement? What do they generateand what does the product bind to and recognized by?
Alternative pathway
generate C3d which binds to Ag and is recognized by CR2 on B cell
TI responses may be facilitated by additional signals derived from microbial products that activate ___ on ___
TLRs on B cells
Some TI non-protein Ags can induce Ig isotypes other than IgM. Which non-protein Ag can induce Ig switch into IgG2?
pneumococcal capsular polysaccharide
Cytokines produced by non-T cells stimulates isotype switching in TI responses. What are these cytokines and which cells produce them?
BAFF produced by DCs and macrophages induces synthesis of AID in Ag activated B cells which is further facilitated by TLRS
TGF-beta mediates the IgA switch and are secreted by non-lymphoid cells at mucosal sites
Are polysaccharides of encapsulated bacteria TI Ags or TD Ags?
TI Ags
What is the major mechanism of host defense against infection by encapsulated bacteria?
Humoral immunity
Individuals with immunodeficiencies of humoral immunity are especially succeptible to life-threatening infections with what specific kinds of Ags/bacteria?
pneumococcus
meningococcus
Haemophilus
TI Ags also stimulate the production of natural Abs which are in circulation and produced without overt exposure to pathogens. What are the characteristics of natural Abs and what cells produce them?
They are low-affinity anti-carbohydrate Abs
produced by peritoneal B-1 cells stimulated by bacteria that colonize in the GI tract and by marginal zone B cells in spleen
Some TI Ags can induce long-lived protective immunity. What class of vaccines can induce this kind of response? What are the characteristics of this type of immunity?
polysaccharide vaccines such as pneumococcal vaccine
Rapid and large secondary responses typical of memory but without much isotype switching or affinity maturation occurs on secondary exposure to these carb Ags
What kind of Abs are anti-ABO Abs and what do they recognize?
They are natural Abs which recognize certain glycolipids (blood group Ags) expressed on the surface of many cell types including blood cells
What are blood group Ags and Abs important for? What are they NOT important for?
important for blood transfusions and transplantation but NOT for host defense
Ag-Ab complexes can simultaneously bind to membrane Ig (through Ag) and the FcgammaRIIB receptor through what part of the Ab?
Fc portion
What is the consequence of the simultaneous binding of the membrane Ig and Fc-gamma-RIIB to the Fc portion?
phosphatases associated with the cytoplasmic tail of the Fc-gamma-RIIB inhibit signaling by the BCR complex and block B cell activation
Regulation of B cell activation by Fc-gamma-RIIB summary
BCR signaling leads to PIP3 formation, which binds other signaling molecules leading to activation–>B cell activation
Fc receptor-associated phosphatase, SHIP converts PIP3 to PIP2 in B cell-receptor complex, blocking downstream signaling–>Block in B cell receptor signaling, no activation
What is FcR-mediated Ab Feedback? What is its purpose?
It is triggered by secreted Ab and blocks further Ab production as opposing the activation via CR2
It’s a physiologic control mechanism in humoral immune response
When do secreted Abs provide complement mediated amplification? When do they provide Fc-receptor mediated inhibition?
Not clear, but hypothesis:
IgM that activates complement but do not bind Fc-gamma-R are involved in amplification
IgG leads to feedback inhibition
In mice whose Fc-gamma-RII was knocked out, what was seen?
Uncontrolled Ab production
Polymorphis in the Fc-gamma-RIIB gene has been linked to susceptibility to what autoimmune disease in humans?
systemic lupus erythematosus
