Download Your lab partner cultured a population of mature B cells in the

BIO331: Immunobiology
Final Exam
Spring 2009
HONOR CODE: CASSANDRA M. SAIKIN
1. Your lab partner cultured a population of mature B cells in the presence of antigen
alone or antigen plus IL-2, IL-4, and IL-5. She is surprised when neither population of B
cells proliferated. You, with your knowledge of B cell activation, can explain why.
Name and describe the missing component (s) and its (their) role in B cell activation.
(20pts)
The cytokines IL-2, IL-4, and IL-5 are not the only players involved in B cell activation.
In fact, they aren’t even the most important. The most important players are the helper T
cells that activate B cells (TH2) by recognizing the presented foreign antigen, as well as
the co-stimulatory molecules CD40 (on the surface of B cells) and CD40L (on the surface
of TH2 cells) in the immune synapse between B and TH2 cells. The activation of B cells
requires several signals from all of these players to help elicit the complete activation of
the lymphocyte. The first signal comes from the binding of antigen to the BCR
(internalization signal). Upon recognition of foreign antigen in the context of self MHC
class II by the T-cell receptor, the TH2 cell is stimulated to express CD40L on its surface,
which then interacts with the CD40 receptor on the surface of the B cell, as well as
secrete the cytokines mentioned above (primarily IL-4). The stimulation of the B cell by
these cytokines alone is not strong enough to activate the B cell. The complete activation
comes from the signal sent from the interaction between the CD40 and CD40L on the Band T-cell surfaces. The signal sent from these surface markers helps to push the B cell
into activation and stimulates important phases in activation, such as B cell proliferation
(pushed into cell cycle), isotype switching (IgM to IgG or IgA) and somatic
hypermutation of the antigen receptor (BCR and antibody) to better recognize the
antigen. They facilitate mutual interaction between the B and helper T cell by inducing
the expression of another co-stimulatory molecule B7, which interacts with CD28 on the
T-cell surface to induce proliferation of T cells.
The lab partner did not have any proliferation of her B cells because she was missing the
TH2 cells as well as CD40L. Therefore, B cell activation will not occur (or will be
extremely weak) in the absence of any of the players mentioned – TH2 cells,
CD40L/CD40 interaction, or cytokines. All of these players work together to drive the
clonal expansion of B cells and have successful humoral response to the antigen.
2. The nude/4 mouse contains B cells and CD8+, but not CD4+ T cells. Describe the
animal's immune response to each of the following and explain your prediction. (10 pts
each)
A. extracellular bacteria – Typically, extracellular bacteria are taken up by B
cells and activated by TH2 cells to produce antibody against it, but this mouse
does not contain helper T cells to perform this function. However, bacteria are
capable of initiating T-independent responses because bacterial polysaccharides
can stimulate B cells in the absence of helper T cells. Therefore, the nude/4
mouse will exhibit a T-independent antigen response that will activate the B cells
and stimulate isotype switching and production of IgG antibody. These
antibodies, as well as IgM antibodies, will opsonize the extracellular bacteria and
can trigger the complement pathway, which will flag the pathogen for
phagocytosis by macrophages/neutrophils (the complement pathway does not
involve activity from CD4 or CD8 cells). The phagocyte will bind the opsonized
bacteria by binding the Fc regions of the antibody opsonin via Fc receptors on the
surface (Fcλ for IgG and Fcµ for IgM) as well as binding C3b of complement via
CR1 complement receptor. This causes receptor cross-linking on the phagocyte
surface, and will allow the pathogen to be engulfed by endocytosis and destroyed.
B. virus – Typically, virus is combated by CD8 cells (cytotoxic T cells). The
nude/4 mouse will most likely exhibit a weak CD8 cytotoxic response because
most CD8 responses require help from CD4 cells. CD8 cells can be activated
strongly by dendritic cells, but only in some situations; more often, CD8 cells are
can only be activated when CD4 cells are bound to the same APC because CD8
cells weakly interact with co-stimulatory molecules. In the nude/4 mouse, the
lack of CD4 cells will result in weak stimulation of CD8 cells, so there will most
likely be few effector CD8 cells in circulation. Therefore, the strongest course of
action to fight virus will come from the NK cells, which have a very similar
effector function to cytotoxic T cells that allows them to fight virally-infected
cells. The NK cells have a completely separate activation process that does not
involve interaction with CD4 cells, so the lack of CD4 cells will have no impact
on the performance of the NK cells.
C. pollen – Pollen is an innocuous agent that stimulates type I hypersensitivity
reactions through IgE activation of mast cells, a histamine-mediated process.
Because IgE production is mediated by TH2 cells that activate B cells and induce
antibody class switching, this will not take place in the nude/4 mouse because of
its lack of CD4 cells. If the nude/4 mouse lacks CD4 cells, there is a chance that
the mouse’s immune system will not mount an allergic response because IgE
antibodies would not be produced and subsequently would not be able to activate
mast cells. At that point, the pollen could be recognized as any other foreign
antigen and mount a normal response; even though the B cells will be able to take
up the pollen, a T-cell dependent response will not be able to produce IgG or IgA
antibodies. It is possible that the pollen could trigger a T-cell independent
response based on its physical properties and produce antibodies, then trigger the
complement system to eliminate the pathogen. Otherwise, the pollen could
trigger persistent infection that will be hard to fight.
3. Can type 1 hypersensitivity be described as antibody or cell-mediated? What cell type
and molecule are central to the second stage of this response? (30 pts)
Type I hypersensitivity is facilitated through mast cell activation, which is an antibodymediated process, mediated by IgE antibodies that propagate allergic responses through
interaction with mast cells, lining the body’s mucosal epithelium. Low doses of
innocuous agent (allergen) that cross the mucosal epithelium get engulfed by antigen
presenting cells (primarily dendritic cells) and secrete the cytokines IL-4 and IL-13 that
favor a TH2 response. The activated TH2 cells then secrete more of these cytokines to
activate B cells and induce isotype switching from IgM to IgE antibody. These resulting
IgE antibodies are specific to the original allergen, and re-exposure to this allergen will
trigger another, stronger allergic response than the previous infection.
Once these allergen-specific IgE antibodies have been selected for, they bind primarily to
the high-affinity FcεRI receptors on the surface of mast cells, and a lesser extent to
basophils and eosinophils. IgE binds to these Fcε receptors without antigen bound to their
receptor, keeping the antibody fixed in the tissues on the mast cells, so as to localize the
allergen and the resulting response. Exposure and binding to the innocuous allergen to
the IgE antibodies causes receptor cross-linking of these IgE-bound Fcε receptors,
resulting in activation of the mast cell. These mast cells contain preformed granules
containing chemical mediators that facilitate the allergic response at the site of exposure.
Activation of these mast cells results in a local inflammation response, and is stimulated
to release its secretory granules within seconds, releasing inflammatory mediators such as
histamine (increases blood flow and vasopermeability), cytokines such as TNF-α that
stimulate expression of adhesion molecules on the surfaces of epithelial cells as well as
vasodilation, and chemokines that recruit eosinophils, basophils, and other lymphocytes
that take part in the allergic reaction. These activated mast cells also express CD40
ligand and IL-4, which can facilitate isotype switching in B cells at the site of exposure,
which amplifies IgE production during a hypersensitivity response.
The location and reactivity of the IgE and mast cells to a particular allergen can facilitate
different responses with different consequences. Normal, regulated reactivity will result
in only localized inflammation which can be overcome easily, while uncontrolled
responses can lead to severe inflammation, causing anaphylaxis and shock, and most
likely death. As noted earlier, repeated exposure to an allergen that has already been seen
will trigger a stronger, faster response the next time around because activated mast cells
with bound IgE are primed and ready to respond at exposure.
4. A hemotologist performed a differential white blood cell count on a patient suspected
of having mononucleosis, neutropenia, or eosinophilia. What would the resulting counts
look like in all three cases? (20pts)
Mononucleosis – Mononucleosis is caused by Epstein-Barr virus (EBV), which infects B
cells and endothelial cells, and is primarily fought by CD8 killer T cells. To a lesser
extent, this virus can be combated with IgG antibody secreted by B cells, which are
activated by TH2 cells. Therefore, we can expect to see an increase in lymphocyte levels,
the most being CD8+ cells, with smaller amounts of CD4+ cells and B lymphocytes.
There would also be an increase in atypical lymphocytes, which are the NK cells, which
are also very good at fighting EBV infection. We would also expect an increase in
macrophages and neutrophils (phagocytes), which would act as garbage cleaners to clean
up the apoptosed cells.
Neutropenia – Neutropenia is defined as an abnormal decrease in circulating neutrophil
levels. It can be caused by numerous types of viral or bacterial infections, as well as
bone marrow deficiencies/disorders. Neutropenia can be correlated with the absence of
leukocytosis (increase in white blood cells). Therefore, in a patient with this condition,
we would expect to see a drop in other leukocytes as well, such as macrophages and B
and T lymphocytes.
Eosinophilia – Eosinophilia is defined as having increased numbers of circulating
eosinophils. The overstimulation of eosinophils can be caused by elevated secretion of
IL-5 by TH2 cells during an infection. Therefore, we would expect to see elevated
eosinophils as well as elevated CD4+ T cells. We would also expect to see increased
levels of other granulocytes, such as mast cells and basophils; chemokines that are
involved in the recruitment of eosinophils (called eotaxins) also recruit these
granulocytes. IgE is also involved in the activation of eosinophils, so we could expect an
elevation in B cell levels with increased levels of eosinophils, which makes sense because
TH2 cells are involved in the activation of B cells and class switching of their antibodies.
If the diagnosis was neutropenia, suggest an infectious disease that would cause such a
change in cell count. (10pts)
X-linked Hyper IgM syndrome is caused by a defect in the CD40 ligand on the surface
of T cells. Typically, during an infection, TH1 cells interact with macrophages through
contact with the CD40 on the surface of the macrophage, stimulating the release of
granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that stimulates
the activation and proliferation of neutrophils (and other granulocytes). With the defect
in CD40 ligand on T cells, there can be no interaction with the macrophage to secrete this
crucial cytokine. Therefore, absence of GM-CSF results in neutropenia because they are
not being selected to proliferate. This will then lead to a prolonged, more dangerous
infection because neutrophils are not present to help fight the cause.
5. In Case Study 17, pg. 101, Tatiana and Alexander were found to have MHC class I
deficiency. When the T cell receptors on the CD8 T cells that the two had were
examined it was found that all were of the  type and not the . How could you
explain this? (look in your textbook for some clues) (20pts)
There are numerous differences between and T cells that can account for the
presence of only cells. During T cell development, cells mature in the periphery
and not in the thymus like cells. This step is determined in early stages of
development when double-negative T cells (CD4- CD8-) are in the process of developing
their T-cell receptors and the signal for chain rearrangements is received first,
promoting the lineage as well as inhibiting the lineage. This commitment and
formation of the TCR then sends these cells to the periphery to continue maturation.
Because these cells do not mature in the thymus, they do not undergo tolerance to self
antigen and self peptide. Therefore, they do not undergo selection to recognize MHC
class I or class II. This makes cells MHC class I independent and are able to
recognize antigen directly or through another class of MHC called MHC class Ib.
Therefore, individuals that have a defect in the TAP1 or TAP2 genes with MHC class I
deficiency can have effective cells; these cells do not utilize the TAP genes because
they are MHC class I independent and do not require the use of the TAP transporter,
which is involved in loading the MHC molecule with the antigen being presented.
6. a. What are the functions of the cell membrane complement receptors? b. What might
be the result of a deficiency in CR1? (30pts)
The complement receptors are located on the surfaces of cells that recognize and bind
pathogen that have been opsonized with different components of complement.
Depending on which type of cell the complement receptor is expressed, this will
determine which type of response is elicited, whether it is phagocytosis, regulating the
complement pathway (protects host from destruction), garbage cleanup, activation of B
cells, or activating G proteins. These receptors can work in conjunction with other
receptors (CRs or not) to propagate a better response.
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CR1 (CD35) – Located on the surfaces of red blood cells, macrophages, and B
cells; on the surface of phagocytes, CR1 recognizes C3b, the main component of
complement, and promotes phagocytosis (this is done in conjunction with the C5a
receptor, otherwise phagocytosis would not occur). CR1 is also involved in
complement regulation, promoting the dissociation of the C3 convertase by
binding to C4b, and can subsequently bind to inhibited C4b (C4bi) and promote
phagocytosis. When CR1 is on the surface of red blood cells, these cells act as
garbage cleaners by engulfing broken-down immune complexes so as not to cause
damage.
CR2 (CD21) – Located on the surfaces of B cells; CR2 is part of the B-cell coreceptor and is involved in complete activation of B cells by recognizing C3d that
has opsonized the pathogen (being recognized by the BCR). It is also capable of
recognized inhibited/broken-down products of complement components (iC3b,
C3dg), as well as Epstein-Barr Virus, which has components homologous to
complement as a mechanism of entry to infect B cells.
CR3 (CD11b/CD18) – Located on the surfaces of macrophages; recognizes
inhibited products of complement (iC3b) and stimulates phagocytosis of the
pathogen.
CR4 (CD11c/CD18) – Located on the surfaces of macrophages; recognizes
inhibited products of complement (iC3b) and stimulates phagocytosis of the
pathogen.
CR1 is the most important complement receptor because it is the primary receptor
involved in the phagocytosis and destruction of complement-coated pathogens; it is the
only receptor that recognizes C3b, the main opsonin. A deficiency in CR1 could result in
impairment in the cleanup of C3b-opsonized pathogens because phagocytes cannot
recognize the pathogen. In the event that these pathogens are not destroyed, the
complement system will initiate late events and the terminal complement components
will be recruited to the pathogen to form the membrane-attack complex, which will
puncture a whole in the wall of the pathogen, destroying it. However, in the absence of
CR1 on the surfaces of erythrocytes, these immune complexes will not be able to be
cleaned up, which can cause organ damage and/or disease. Therefore, a deficiency in
CR1 could result in systemic damage to the host.
7. a. What is the relative importance of granzymes, perforins, and lymphotoxins in T cell
cytotoxicity? b. Why might 3 distinct mechanisms of cytotoxicity have evolved in a
single cell type? (30pts)
Granzymes, perforins, and lymphotoxins (TNF-β) are all primary components of T cell
cytotoxicity that stimulate apoptosis of virally-infected cells. Perforin and granzymes
work together to penetrate and enter the infected cell and activate apoptotic proteins,
while lymphotoxin stimulates apoptosis through surface receptor interaction. All of these
players act (either directly or indirectly) to activate the caspase proteins that facilitate
apoptosis. Granzymes and perforin (cytotoxic proteins) are secreted together in their
active forms from cytotoxic granules by cytotoxic T cells in the direction of the virallyinfected cell (due to polarization of Golgi apparatus). Perforin acts to deliver the
granzymes into the cytoplasm by punching holes in the surface of the cell to allow the
granzymes to enter the cell. These granzymes, which are serine proteases, activate
caspases by proteolytic cleavage (pro-caspase is inactive). Perforin can also degrade
some cytosolic proteins. In contrast, lymphotoxin (a cytokine), which is secreted as a
homotrimer, binds to the receptor TNFR-1 on the surface of the infected cell, causing
trimerization of these receptors and inducing apoptosis via death domains. Interaction
between recruited death domains then stimulates the activation of pro-caspases via
proteolytic cleavage. These caspases (activated by lymphotoxin or granzymes) then
activate CAD (via cleavage), which then enters the nucleus and cleaves DNA, which
completely kills the infected cell.
It is likely that these three mechanisms of cell-mediated apoptosis work together in
cytotoxic T cells in order to facilitate a more efficient cytotoxic response and ensure that
the infected cell is killed. The presence of these three mechanisms ensures that the
infected cell receives at least one apoptotic signal, so in the case that one mechanism is
impaired or weak, the other will be there to facilitate apoptosis.
8. Construct a hypothetical pathogen/antigen that might selectively stimulate a Th1
response. How would you test this activity? (20pts)
An example of a pathogen that stimulates a TH1 response over a TH2 response would be
Mycobacterium leprae, a bacillus that colonizes in macrophages and is responsible for
Tuberculoid Leprosy. A TH1 response is favored because the mycobacterium can
stimulate the infected macrophage to secrete IL-12, which favors the differentiation of
TH0 cells into TH1 over TH2 cells in the early stages of the response. These selected TH1
cells then secrete IL-2, IFN-λ, and TNF-β to have continual activation of macrophages
and proliferation of TH1 cells, as well as sequestering of TH2 cells. IFN-λ is the primary
player involved in the activation of macrophages to kill the colonized M. leprae, as well
as block the production of TH2 cells. In this form of leprosy, the pathogen is present at
low to undetectable levels and has a low infectivity rate because of the vigorous cell-
mediated response that is able to contain and stop the spread of infection (although it is
not completely eradicated). The presence of only TH1 cells allows for the continued
activation of infected macrophages to kill the bacteria present in its endosomes. This
results in local inflammation only because the infection is contained and does not become
systemic.
To test the activity of an active TH1 (cell-mediated) response, a delayed-type
hypersensitivity reaction is performed. This reaction tests for exposure to the
mycobacterium by mediating an effector TH1 response by injecting antigen specific to M.
leprae. The effector TH1 cells (that have already seen antigen) enter the site of injection,
recognize the antigen in context of self MHC class II, and then secrete the inflammatory
cytokines IFN-λ (activates macrophages) and TGF-β. These cytokines facilitate
vasodilation and vasopermeability to increase the influx of recruited leukocytes, causing
swelling and redness, which is characteristic of a positive delayed-type hypersensitivity
response. This proves that there is normal T-cell responsiveness and indicates a M.
leprae infection.
Bonus: (10pts) Define the single most important thing you learned about immuno!
The most important thing I learned is that nothing in immunology is ever simple –
nothing ever occurs in a vacuum and the majority of reactions take multiple hits to have a
complete and accurate response. This is true in the case of B- and T-cell activation,
which requires multiple signals for complete activation. For example complete B-cell
activation requires not only internalization of antigen (upon binding to BCR) and
recognition by TH2 cells, but it requires signals sent by co-stimulatory molecules
(CD40/CD40L) as well as cytokines sent by the T cell in order for the B cell to receive
the complete activation signal. Another example is the binding of antigen to B or T cells
– this initial signal is strongest when antigen-binding causes cross-linking of the antigen
receptors.