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Lecture 13: The Humoral Immune Response, Pt 1

Terms in this set (108)

B cells =
humoral immunity
What is the role of B cells?
produce antibodies
What do antibodies do?
-neutralize
-opsonize
-activate complement
What activates B cells?
-Helper T cells (thymus dependent)
-Antigen (thymus independent)
What does B cell activation start with?
B cell receptor
BCR can:
1. initiate signal cascade
2.Deliver antigen to intracellular sites for antigen processing, so antigenic peptides bound to MHC class II molecules can be returned to the surface
What is the first signal?
this initial binding with an antigen
Antigens (1st signal)
-the naive B cells will encounter antigens in the lymph nodes
How do antigens move to the lymph nodes?
Via lymphatic system in 2 ways:

opsonized antigens bearing C3b are trapped by specific receptors

opsonized antigens are taken up by specialized macrophages, which present the antigens to B cells (this also allows direct transport of the antigen to the follicle to the dendritic cells that reside there)
What else is required to activate a B cell?
a second signal
-TI or TD antigens
Thymus-independent (TI) antigens
can activate B cells without any help from T cells
Thymus-dependent (TD) antigens
can only activate B cells when presented by Helper T cells
TI activation
-some microbes produce antigens that can induce antibody responses with no help from T lymphocytes
What typically occurs to cause TI activation?
when the 1st signal is with the BCR and the 2nd signal is through TLR signaling
-often with microbial antigens that are highly repetitive, such as LPS
What does the TI antigen activate?
the NFkB pathway
What things need to happen for TD antigens?
-CD4 T cells specifically the T follicular Helper Cells (T FH) need to undergo activation after binding to an APC
-these cells proliferate and are ready to interact with naive B cells
Continuing TD antigen responses
B cells must come into contact with antigen and process it. They will display MHCII:antigen complex

If it matches to the T (FH) then this 2nd signal will cause the B cell to undergo the last steps of maturation

also activates the NFkB pathway
What are the relevant proteins involved in the signaling for TD antigen responses?
-TCR + CD3 binds to MHCII:antigen
-Co-receptor CD40 on B cell will bind to ligand on T cell (in this case CD40L)
Once bound what 2 main things occur to the B cell?
1. T (FH) cell provides B cells with signals to promote survival

2. T (FH) cell provides B cells with signals to promote proliferation
In TD antigen responses the same antigen recognized by B cells must be:
the same antigen that activates T cells which is called linked recognition
Why is a random encounter inefficient?
the frequency of a naive lymphocyte for a particular antigen is very low (< 1 in 10,000)

when you combine the fact that both the T (FH) cell and the B cell have to have the same antigen the likelihood is less than 1 in 100,000,000
So how can this work?
these cells express receptors that take them right to the lymph nodes where they interact
Both B and T cells leave the lymphatic tissue by expression of what?
sphingosine 1-phosphate receptor (S1P receptor) which follows the gradient of S1P
How do T cells enter the T-cell areas of the lymphoid follicles?
by expressing CCR7
Circulating naive B cells express what to get to the right place at the right time and what does it bind to?
CXCR5
-which is a receptor that binds to the chemokine CXCL13
CXCL13
abundant in the primary lymphoid follicle
Once a naive B cell is in the follicle what happens?
survival is promoted by BAFF (B cell activating factor)

and the naive B cell expresses EB12 receptor
What does the EB12 receptor do?
moves the cell toward the outer follicles (where the antigens are entering)
How long does the B cell sample antigens and after what does it do?
6 hours to 1 day
-the B cell induces expression of CCR7
At the same time of B cells inducing expression of CCR7 what are activated T cells doing?
expressing CXCR5 which will move them toward the follicle
This puts B and T cells in:
the same time and space to increase the likelihood of meeting
What happens to B cells after an initial encounter with a T cell?
they reduce expression of CCR7 and begin to migrate away from the border due to increase of expression of EB12

proliferation and differentiation of B cells begins
Activated B cells
are the B cells that have received the 1st signal and are no longer naive B cells, but have also not completed development
Activated (resting) B cells either form a:
-primary focus
-germinal center
Primary focus
the B cells that are expressing the EB12 after encountering T cells begin to aggregate and differentiate in the primary focus

primary foci can begin to form at 5 days post exposure

this proliferation and differentiation at the primary focus is the first phase of the primary humoral response
some of the resting B cells at the primary focus will differentiate into what?
plasmablasts
Plasmablasts
synthesize antibodies with low-specificity BUT are fast

they are short-lived and can serve as a stop gap while antibodies with higher affinity for the specific antigen are being produced

mostly produce IgM pentamers
Germinal centers
these centers are mainly differentiating B cells, but about 10% of the cells are T(FH) cells to help direct B cell differentiation

formation of germinal centers begins the 2nd phase of the humoral immune response

primarily produces plasma cells
Plasma cells
synthesize antibodies with very high specificity to an antigen but take much longer than plasmablasts

also longer lived

plasma cells=antibody secreting cells
How do we get plasma cells with high specificity?
in the germinal centers, plasma cells are created to have very high specificity to the antigen using 3 processes:
-somatic hypermutation
-affinity maturation
-class switching
Somatic hypermutation
process that introduces mutations into the V regions of immunoglobulin genes

initiates affinity maturation

produces very closely related B cell clones that differ subtly in specificity and antigen affinity
Enzyme AID
process of mutation is initiated by an enzyme called activation-induced cytidine deaminase (AID)

this process creates mutations in the V-region genes at a rate of about one bp change per 10^3 base pairs per cell division

background mutation is about one bp change per 10^10 bps per cell division
How does enzyme AID work?
it created point mutations by deanimating cytidine to form uridine

the occurs only when the DNA strand is unwound during transcription for proliferating cells in the germinal center

somatic hypermutation cannot occur in areas that are not being actively transcribed

AID can mutate other transcribed genes in B cells but at a much lower rate
Why is the presence of uridine in place of cytosine problematic?
1. it is foreign to normal DNA

2. there is a mismatch to the guanosine (what cytosine normally binds to)
What do these problems trigger?
several kinds of DNA repair including:
-mismatch repair
-base-excision repair
What does "Fixing" the problem do?
it causes more alterations to the DNA sequence
mismatch repair
proteins remove the problematic uridine along with several adjacent nucleotides

DNA polymerase then "patches" the damaged strand by filling in the nucleotides

this often causes mutations at A:T base pairs

it is highly error prone
base excision repair
the enzyme uracil-DNA glycosylase (UNG) removes uracil base from the uridine to create an abasic site

during the next round of DNA rep, a random nucleotide opposite will be inserted by a DNA polymerase

this most often leads to mutations at C:G bps

it can also lead to the action of another enzyme: APE1
APE1
at the abasic site, the enzyme apurinic/apyrimidinic endonuclease 1 (APE1) can remove the abasic residue to create a single strand nick in the DNA

this can result in gene conversion

important in some mammals (not humans) and birds
These actions lead to:
isotype switching
isotypes are defined by
the constant region of an Ig
in humans there are 5 isotypes:
IgG, IgA, IgE, IgM,
IgD (only on naive B cells)
When does isotype switching occur?
only after B cells have been stimulated by an antigen and only in germinal centers
steps of isotype switching
repetitive DNA known as switch regions lie in the introns b/w the J(H) gene segments and the Cu gene and equivalent sites for other heavy-chain isotypes

AID -> UNG -> APE1 causes single-strand nicks in the DNA at random locations at the switch regions

repair proteins act to initiate repair of the double strand

the repair machinery will join 2 switch regions and excise the intervening sequences

the selected constant region (which equals a different isotype) is now adjacent to the VDJ region
How do T (FH) cells direct the right choice of isotype for infection?
by secreting appropriate cytokines
-see chart on slide 36
affinity maturation
ensures the the developing cells with the highest affinity to the antigen are selected
the anatomy of the germinal centers (GCs) is important to understand what?
affinity maturation
What are the 2 zones of GC?
light and dark zones
-as B cells mature and undergo selection they move through these zones controlled by expression of different receptors
What small class of dendritic cells are in the light zone of GCs in the follicles of lymph nodes?
follicular dendritic cells (FDCs)
follicular dendritic cells (FDCs)
sole purpose is to continually present the antigen to developing B cells

these FDCs store the antigens as immune complexes on their surface
Affinity and specificity are continually refined through the:
cyclic reentry model
Cyclic reentry model
1. B cell has slight mutations due to somatic hypermutation in the dark zone

2. competes for the antigens on the FDCs in the light zone

3. binds to the T(FH) cells through MHC II in the light zone

4. receives signals to continue refinement in the dark zone
Positive selection
B cells that express receptors with higher affinity to the antigen will be more likely to bind to that antigen over the slightly different receptors

this allows them to present the antigen with MHC II receptors to the T(FH) cells

the T(FH) cells give these cells survival and mitogenic signals via CD40 and cytokines

the surviving cells reenter the dark zone to improve even more
Most mutations will:
have a negative effect on the ability of a B cell to bind to the antigen, and it will be selected against and eliminated by apoptosis

a special type of macrophage proliferated in GCs to quickly eliminate B cells with negative mutations
Eventually the developing B cells will leave the germinal center and differentiated into:
plasma cells or memory cells
Most plasma cells will:
circulate to the site of infection where they will secrete copious amounts of antibody

some plasma cells will migrate to the bone marrow, where they will survive for long periods of time
memory cells:
do not secrete antibodies and live for a very long time
IgG subclasses
IgG1, IgG2, IgG3, IgG4
-named by decreasing order of abundance in serum
IgA subclasses
IgA1 and IgA2
the different heavy chains that make up the isotype are named using what?
the lower case Greek letter that corresponds with the isotype
IgM
μ
IgD
δ
IgG
γ
IgE
ε
IgA
α
IgM
-first class of Ig to be produced after activation
-Low affinity
-Pentamer - this increases avidity
-in blood stream
-can activate complement component C1
IgG
-in extracellular space and blood
-activate complement component C1
IgD
-signals to B cell to activate
-role is poorly understood
IgA
-bloodstream
-mucosal surfaces, gut, respiratory tract
-mother's breast milk
-cannot activate complement
IgE
-involved in defense of extracellular parasites
-allergies
-cannot activate complement
How do antibodies secreted by plasma cells get to the right location?
there are specific receptors that bind to specific antibodies to help transport them
Polymeric immunoglobulin receptor (plgR)
helps to transport IgA and IgM antibodies from the gut lumen to the gut epithelium
Why are newborns very vulnerable to infection?
because they have no previous exposure to pathogens
What antibodies pass through maternal milk?
IgA
What antibody is transferred across the placenta directly to the fetus and how?
IgG through the neonatal Fc receptor (FcRn)
Where else are FcRns located in adults?
the gut, liver, and endothelial cells
We have gotten the super specific antibodies to the right location - now what?
depending upon their specific isotype, antibodies will work to neutralize, opsonize, and/or activate complement
What antibodies are good at neutralization?
High affinity IgG and IgA antibodies are especially good at neutralization to prevent other cells from being harmed by the pathogen
What is the result of all of these antibodies binding to antigens?
immune complexes form
immune complexes
typically small clusters of antigen:antibody complexes that have bound during neutralization

can be cleared by utilizing the Fc region of the antibody

phagocytic cells express receptors for Fc and bind to the exposed ends of antibodies
How do we kill the neutralized pathogens?
our immune system utilized the Fc regions by having a variety of accessory effector cells bear Fc recpetors
What phagocytic cells bear Fc receptors?
macrophages
dendritic cells
neutrophils
What non-phagocytic cells bear Fc receptors?
NK cells
Eosinophils
Basophils
Mast cells
Fc receptors facilitate what?
an antigen specific attack using a cell that is not antigen specific
What are the most important Fc receptor bearing cells?
the phagocytes that ingest and destroy bacteria (like macrophages and neutrophils)

many bacteria can be detected by phagocytes but not all

these pathogens become susceptible when coated with antibodies that will engage the Fc receptors
What other events can occur through the activation of macrophages?
inflammation
Can free floating antibodies activate macrophages?
no, only when the Fc receptors are cross-linked will the macrophage become activated

successive binding of these receptors aids in engulfing the pathogen through this mechanism
How are NK cells activated by antibodies?
by process called antibody-dependent cell-mediated cytotoxicity (ADCC)
What Fc do NK cells recognize?
they have Fc receptors that recognize the Fc from IgG1 and IgG3 subclasses

NK cells release cytotoxic granules when there is binding to these Fc receptors
Mast cells
are myeloid cells that reside in the tissues
Basophils
are myeloid cells that reside in the blood stream
Mast cells and basophils
can release a mix of chemical mediators including histamine, that act to make the blood vessels more permeable
What Fc do mast cells have receptors for?
IgE and IgG
What happens when mast cells are activated?
they release lipid inflammatory mediators and cytokines

unlike other Fc receptors Mast cells have highly specific receptors for IgE and will stably bind but cross linking is still required for activation
When is histamine released?
when the mast cell is activated
Histamine
is an important initiator of inflammation

mast cells are also responsible for many allergic responses
parasites
there are multiple ways to deal with parasites which are typically too large to simply be engulfed

1. macrophages can attach to the outside and release toxic granules

2. IgE can activate the role of mast cells

3. IgE can activate the eosinophils
Macrophage activity at the parasites
to activate the surface of the pathogen is coated with antibodies (typically IgA, IgG, or IgE)

this attracts specific Fc receptors that will often use exocytosis to expose the parasite to toxic proteins
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