Test Bank for Janeway’s Immunobiology 9th Edition

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 JANEWAY'S IMMUNOBIOLOGY, 9TH EDITION
CHAPTER 6: ANTIGEN PRESENTATION TO T LYMPHOCYTES
The generation of : T-cell receptor ligands
6-1 Antigen presentation functions both in arming effector T cells and in triggering their
effector functions to attack pathogen-infected cells

6.1 Matching: The diagram in Figure Q6.1 shows a pathogen (in red) that is present in
different cellular compartments of each of the cell types shown. In each case, a specific
T cell subset will recognize peptides of that pathogen presented on MHC molecules on
the surface of the cell, and will execute its effector function. From the list below, match
the appropriate T cell effector response to the cell type and location of the pathogen.

Figure Q6.1
i. CD4 T cell killing of target cell
ii. CD8 T cell killing of target cell
iii. CD4 T cell activation of target cell’s antibody production
iv. CD8 T cell activation of target cell’s antibody production
v. CD4 T cell activation of target cell’s ability to kill intracellular pathogen
vi. CD8 T cell activation of target cell’s ability to kill intracellular pathogen

6.2 Multiple choice: The mechanism of cross-presentation by dendritic cells is an essential

pathway for generating CD8 T cell responses to some intracellular pathogens. If this
pathway did not exist, we would be highly susceptible to:
A. Intracellular pathogens that can survive inside macrophage endocytic vesicles
B. Intracellular pathogens that are able to evade antibody responses
C. Intracellular pathogens that do not infect and replicate in dendritic cells
D. Intracellular pathogens that can spread from cell to cell by inducing cell fusion
E. Intracellular pathogens that infect and replicate in red blood cells

6-2 Peptides are generated from ubiquitinated proteins in the cytosol by the proteasome

6.3 Multiple choice: The adaptive immune system developed a strategy for monitoring the
proteins synthesized in virtually any cell in the body, thereby preventing pathogens from
‘hiding out’ by adopting an intracellular lifestyle. To accomplish this, the immune system:
A. Co-opted the ubiquitin-proteasome system used by cell for protein turnover
B. Created a novel pathway using the immunoproteasome for generating peptides
C. Created a novel pathway to express foreign proteins on the cell surface
D. Took advantage of proteolytic enzymes present in endocytic vesicles
E. Engineered an immune-specific ubiquitin molecule for tagging foreign proteins

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6.4 Multiple choice: Virus infections induce production of interferons that act on infected
cells to enhance their recognition by CD8 cytotoxic T cells. To counter these
mechanisms, viruses often encode proteins that interfere with antigen processing and
presentation. In an experiment, cells infected with Virus X are treated with interferon and
compared with uninfected cells treated with interferon. Proteasomes are isolated from
the two cell populations and their enzymatic activities are compared. The data in Figure
Q6.4 show the amino acid preferences for cleavage of peptides by the two samples of
proteasomes.

Figure Q6.4
Based on these data, Virus X most likely encodes a protein that interferes with:
A. The expression of MHC class I on the surface of the infected cell
B. The rate at which peptides are produced from intact proteins in the infected cell
C. The transport of peptides from the cytosol to the endoplasmic reticulum in the
infected cell
D. The replacement of constitutive proteasome subunits with immunoproteasome
subunits in the infected cell
E. The development of CD8 T cells in the thymus by inhibiting the
thymoproteasome

6-3 Peptides from the cytosol are transported by TAP into the endoplasmic reticulum
and further processed before binding to MHC class I molecules

6.5 Short answer: A cell line carrying a mutation in a single gene is found to express very
low levels of MHC class I on its surface. When infected with influenza virus, these cells
are not recognized nor are they killed by a CD8 T cell line specific for an influenza
peptide bound to the MHC class I protein expressed by these cells. Incubation of the
mutant cell line with a large excess of this peptide in the cell culture medium overnight
leads to the results shown in Figure Q6.5.

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 Figure Q6.5

What is the most likely candidate for the gene that is defective in the mutant cell line?

6-4 Newly synthesized MHC class I molecules are retained in the endoplasmic reticulum
until they bind a peptide

6.6 Multiple choice: During MHC class I synthesis and folding in the endoplasmic reticulum
(ER), a process of peptide editing takes place as the newly synthesized MHC class I
protein is held in a ‘peptide receptive’ state by binding to the calreticulin:ERp57:tapasin
complex. Peptide editing ensures that the MHC class I molecules that reach the cell
surface have stable, high affinity binding for their peptide cargo. Peptide editing is
important to the immune response because it:
A. Maintains high levels of surface MHC class I expression
B. Ensures that MHC class I molecules are not degraded in the ER
C. Retains the nascent MHC class I molecule in a peptide receptive state
D. Allows surface MHC class I molecules to bind new peptides from the extracellular
milieu
E. Prevents surface MHC class I molecules from undergoing peptide exchange at
the cell surface

6.7 True/False: MHC class I surface expression is dependent on an abundant source of

pathogen-derived peptides. Thus, in uninfected cells, nearly all of the MHC class I
proteins are degraded and never reach the cell surface.

6-5 Dendritic cells use cross-presentation to present exogenous proteins on MHC class
molecules to prime CD8 T cells

6.8 Multiple choice: The virus shown in the diagram below is only able to infect and
replicate in epithelial cells. In order for the cross-presenting dendritic cell to display viral
peptides, rather than self peptides on its surface MHC class I proteins, which of the

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 following procedures could be utilized, starting with the components shown in Figure
Q6.8?

Figure Q6.8
A. Mix epithelial cells with heat-killed virus, wait 24 hrs, wash away any virus
particles outside the epithelial cells, then add epithelial cells to dendritic cells.
B. Mix epithelial cells with viral peptides, wait 24 hrs, wash away any viral peptides
not bound to the epithelial cells, then add epithelial cells to dendritic cells.
C. Mix epithelial cells with live virus particles, wait 24 hrs, wash away any virus
particles outside the epithelial cells, then add epithelial cells to dendritic cells.
D. Mix dendritic cells with viral nucleic acids and epithelial cells for 24 hrs.
E. MIx epithelial cells will viral nucleic acids, wait 24 hrs, wash away any viral
nucleic acid remaining outside the epithelial cells, then add epithelial cells to
dendritic cells.

6.9 Multiple choice: Some viruses have mechanisms to down-regulate MHC class I protein
expression on the surface of cells in which the virus is replicating. This immune evasion
strategy might prevent effector CD8 cytotoxic T cells from recognizing and killing the
virus-infected cells. Would this immune evasion strategy also prevent the initial
activation of virus-specific CD8 T cells?
A. Yes, because no viral peptide:MHC class I complexes would form to activate
CD8 T cells.
B. No, because dendritic cells would take up infected cells and cross-present viral
peptides to activate CD8 T cells.
C. No, because some presentation of MHC class I complexes with viral peptides
would occur before the virus could down-regulate all the surface MHC class I
protein.
D. Yes, because this immune evasion strategy would also function in dendritic cells,
even if the virus doesn’t replicate in dendritic cells.
E. No, because the type I interferon response induced by the virus infection will up-
regulate MHC class I expression and override the immune evasion mechanism.

6-6 Peptide:MHC class II complexes are generated in acidified endocytic vesicles from
proteins obtained through endocytosis, phagocytosis, and autophagy

6.10 Multiple choice: Three major cell types, dendritic cells, macrophages, and B cells,
present peptides bound to MHC class II molecules for recognition by CD4 T cells. In
general, these peptides are derived from proteins or pathogens taken up by the cell by
endocytosis, phagocytosis, or macropinocytosis. Based on these pathways of antigen
uptake, some of the enzymes that degrade proteins to generate peptides for MHC class
II presentation are:

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 A. Ubiquitin ligases that tag proteins for degradation by the proteasome
B. ATP transporter proteins that deliver endocytic proteins into the cytosol for
degradation
C. Cysteine proteases like cathepsins that function at acidic pH
D. The lysosomal thiol reductase found in the endosomes
E. The lysosome-associated membrane trafficking protein, LAMP-2

6-7 The invariant chain directs newly synthesized MHC class II molecules to acidified
intracellular vesicles

6.11 True/False: The invariant chain protein, Ii, has only one function in MHC class II antigen
presentation. This function entails Ii protein occupying the peptide-binding site of each
newly synthesized class II protein, thereby preventing nascent MHC class II proteins
from binding peptides or misfolded proteins in the endoplasmic reticulum.

6-8 The MHC class II-like molecules HLA-DM and HLA-DO regulate exchange of CLIP for
other peptides

6.12 Multiple choice: Peptide editing is an important component of antigen presentation for
both MHC class I and MHC class II pathways, as it drives the preferential presentation of
high-affinity binding peptides. For MHC class II peptide editing, HLA-DM plays a key
role. In the absence of HLA-DM:
A. MHC class II molecules traffic to the cell surface with CLIP in their binding sites.
B. No MHC class II molecules are released to traffic to the cell surface.
C. MHC class II molecules bind to HLA-DO and are inhibited from binding peptides.
D. Pathogens can evade the immune system by blocking peptide exchange on
MHC class II.
E. HLA-DO competes for high-affinity binding peptides with MHC class II molecules
and blocks antigen presentation.

6.13 Multiple choice: Empty MHC class I and MHC class II molecules are rapidly removed
from the cell surface. This process prevents:
A. The accumulation of empty MHC molecules on the cell surface which would
interfere with T cells recognizing pathogen-derived peptide:MHC complexes
B. Pathogens from evading the immune response by inducing peptide release from
cell surface MHC molecules
C. MHC class I molecules from being internalized into endosomes and binding
endosome-derived peptides
D. HLA-DM from trafficking to the cell surface with MHC class II
E. Inappropriate T cell recognition of healthy cells that are not infected, nor have
ingested a pathogen

6-9 Cessation of antigen processing occurs in dendritic cells after their activation
through reduced expression of the MARCH-1 E3 ligase

6.14 Multiple choice: The MARCH-1 E3-ubiquitin ligase is expressed in B cells, dendritic
cells, and macrophages. The pathway regulated by MARCH-1 is targeted by some
pathogens in an immune evasion strategy. In this strategy, the pathogens encode:
A. A protein that induces degradation of MARCH-1
B. A protein that mimics MARCH-1 and functions similarly
C. A protein that binds to MARCH-1 and inhibits its function

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 D. A protein that is induced by IL-10 in macrophages and dendritic cells
E. A protein that induces degradation of CD86

The major histocompatibility complex and its function

6-10 Many proteins involved in antigen processing and presentation are encoded by
genes within the MHC

6.15 True/False: The genes encoding MHC proteins are closely linked with genes encoding
proteins involved in antigen processing and presentation. This genetic linkage facilitates
the coordinate regulation of these genes by interferons.

6-11 The protein products of MHC class I and class II genes are highly polymorphic

6.16 Multiple choice: The adaptive immune system uses multiple strategies to generate
diversity in our ability to mount responses to a wide array of infectious microorganisms.
These strategies include the generation of diverse repertoires of B-cell and T-cell
antigen receptors, as well as polymorphism of MHC genes. The polymorphism of MHC
genes differs from the diversity of lymphocyte antigen receptors in that:
A. It involves DNA rearrangements at multiple gene segments in the MHC locus.
B. It requires different enzymes than the RAG1/RAG2 recombinase required for
antigen receptor rearrangements.
C. It results in a diverse repertoire of clonally distributed receptors on dendritic cells,
rather than on lymphocytes.
D. It creates diversity between individuals in the population rather than within a
single individual.
E. It does not contribute to the transplant rejection responses that occur after organ
transplantation between unrelated individuals.

6.17 Short answer: Multiple mechanisms contribute to create a wide diversity in MHC protein
expression between different individuals in the population. In addition to the genetic
polymorphism of MHC genes, what additional mechanism(s) contribute to this diversity?

6-12 MHC polymorphism affects antigen recognition by T cells by influencing both

peptide binding and the contacts between T-cell receptor and MHC molecule

6.18 Multiple choice: MHC polymorphism at individual MHC genes appears to have been
strongly selected by evolutionary pressures. In other words, there appears to be
selection for maintaining hundreds to thousands of different alleles of each MHC gene in
the population. This notion is based on the observation that nucleotide differences
between alleles that lead to amino acid substitutions are more frequent than those that
are silent substitutions (i.e., not changing the amino acid sequence of the protein). In
addition, the positions within the MHC protein where most of the allelic sequence
variation occurs are not randomly distributed, but are concentrated in certain regions of
the MHC protein. This latter point indicates:
A. That some nucleotide sequences within the MHC genes are hot-spots for
mutation
B. That MHC genes are more susceptible to point mutations than to larger
nucleotide deletions

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 C. That MHC allelic polymorphism has been driven by selection for diversity in
peptide binding specificity
D. That MHC genes are more susceptible to all types of mutations than are other
genes in the genome
E. That MHC polymorphism has evolved to prevent pathogens that infect non-
human primates from infecting humans

6.19 Multiple choice: The experiment shown in Figure Q6.19 uses two strains of mice that
differ in their MHC genes. Strain A is H-2a and Strain B is H-2b. Mice of each strain are
infected with the virus LCMV, and T cells are isolated at day 8 post-infection. These T
cells are mixed with target cells that express either H-2a or H-2b; in each case, the target
cells are either uninfected or infected with LCMV. After a four-hour incubation of T cells
with target cells, the percentage of target cells lysed by the T cells is shown in the graph.

Page 7 of 22
 Figure Q6.19
The explanation for the results of this experiment is:
A. Mice of strain B do not make a T cell response to LCMV.
B. Mice of strain A make a more robust T cell response to LCMV than mice of strain
B.
C. Target cells that express H-2b cannot be infected with LCMV.
D. T cells from mice of strain A only recognize viral peptides on target cells
expressing H-2a.
E. LCMV peptides do not bind to MHC class I molecules from H-2b mice.

6-13 Alloreactive T cells recognizing nonself MHC molecules are very abundant

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6.20 Multiple choice: In a mixed lymphocyte reaction, T cells from individual A make a
robust response to antigen-presenting-cells from individual B, as long as the two
individuals express different alleles of MHC molecules. Estimates indicate that up to
10% of the T cells from individual A may contribute to this response. If one performed
this assay using responder T cells from a child and antigen-presenting cells from one
parent, the result would be:
A. A massive proliferative response made by the antigen-presenting cells of the
parent
B. A very weak response by the child’s T cells, involving only 0.1% of their T cells
C. The complete absence of any proliferative response by the child’s T cells
D. A robust cytolytic response that kills all of the parent’s antigen-presenting cells
E. A robust response by the child’s T cells

6.21 True/False: Alloreactivity refers to the ability of T cells to respond to allelic

polymorphisms in MHC molecules when mixed with antigen-presenting cells from a
genetically different individual. The T-cell receptors involved in alloreactive responses
are recognizing amino acid sequences on foreign MHC molecules and do not interact at
all with the peptides bound to these MHC molecules.

6-14 Many T cells respond to superantigens

6.22 Multiple choice: Several types of pathogens encode proteins that function as
superantigens, which activate massive numbers of T cells in an individual. One example
is the staphylococcal enterotoxins that cause food poisoning. These superantigens are
the exception to the general rule that T cells only recognize specific peptide:MHC
complexes, because they:
A. Induce activation of any T cell whose T-cell receptor uses a particular Vβ region
bound by that superantigen
B. Simultaneously stimulate all of the T-cell receptors on a given T cell
C. Cover up the peptide-binding site, preventing MHC molecules from binding
peptides
D. Activate a large number of T cells that are specifically recognizing peptides
derived from the superantigen protein
E. Induce anti-microbial cytokine production that aids the immune system in clearing
the pathogen

6-15 MHC polymorphism extends the range of antigens to which the immune system can
respond.

6.23 Multiple choice: The extensive polymorphism of MHC genes in the population is
thought to represent an evolutionary response to outflank the evasive strategies of
pathogens. This polymorphism makes it difficult for pathogens to eliminate all potential
MHC binding epitopes from their proteins. Based on this reasoning, it would seem
advantageous for each individual to encode more than three different MHC class I and
three different MHC class II genes per chromosome copy. If some individuals in the
population had MHC loci that encoded 10 different MHC class I and 10 different MHC
class II genes, the T cell repertoire in those individuals would likely be:
A. Much more diverse than in the rest of the individuals of that population
B. Much better at recognizing rare pathogens not encountered by most individuals
in that population
C. Much less diverse than the rest of the individuals in that population

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 D. Much more alloreactive than the T cells found in the other individuals of that
population
E. Very reactive to bacterial and viral superantigens

Generation of ligands for unconventional T-cell subsets

6-16 A variety of genes with specialized functions in immunity are also encoded in the
MHC

6.24 True/False: The MHC locus encodes a large number of genes spanning over four
million bp of DNA. These include many genes involved in antigen processing and
presentation, as well as receptors recognized by non-conventional T cells and natural
killer (NK) cells. In addition, the MHC locus encodes genes with no function in immunity
at all.

6-17 Specialized MHC class I molecules act as ligands for the activation and inhibition of
NK cells and unconventional T-cell subsets

6.25 Multiple choice: NKG2D is an activating receptor expressed on NK cells, : T cells,
and some cytotoxic : T cells. When stressed or infected cells up-regulate receptors
that bind to and activate NKG2D molecules, the stressed or infected cells will be killed.
This pathway relies on the fact that stressed or infected cells up-regulate:
A. All classical MHC class I molecules
B. HLA-C molecules that bind KIRs
C. MHC class Ib genes such as MICA, MICB, and RAET1
D. Qa-1 and HLA-E molecules that bind leader peptides of other HLA class I
molecules
E. HLA-G molecules just like those expressed on the fetal-derived cells in the
placenta

6-18 Members of the CD1 family of MHC class I-like molecules present microbial lipids to
invariant NKT cells

6.26 Multiple choice: Some CD1 molecules bind to glycosphingolipids, and are recognized
by a subset of T cells known as invariant NKT (iNKT) cells. The ability of these T cells to
recognize different glycolipid constituents from microorganisms when they are bound to
CD1d places these cells in the ‘innate immune’ category. While iNKT cells do express a
fully rearranged : T-cell receptor, one key feature of the T-cell receptors expressed on
iNKT cells also places them in the ‘innate immune’ category. This feature is:
A. iNKT cells have a highly restricted T-cell receptor repertoire, with the majority of
cells utilizing the same V and J rearrangement.
B. iNKT cells express receptors that are also expressed on NK cells.
C. iNKT cells express T-cell receptors that induce inhibitory, rather than activating
signals.
D. iNKT cells do not generally express CD4 or CD8.
E. The T-cell receptors expressed on iNKT cells recognize both MHC class I and
MHC class II molecules.

6-19 The nonclassical MHC class I molecule MR1 presents microbial folate metabolites
to MAIT cells

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6.27 Short answer: NKT cells that recognize microbial glycolipids bound to CD1 molecules
comprise a class of T cells that shares features of both innate and adaptive immune
cells. A second class of such cells are MAIT cells, that recognize antigens bound to the
MHC class Ib molecule, MR1. What is the class of PAMP recognized by MAIT cells?

6-20 : T cells can recognize a variety of diverse ligands

6.28 Multiple choice: T cells expressing : T-cell receptors have been found to recognize a
diversity of ligands, including pathogen-derived proteins, self-peptides, and stress-
induced molecules. This pattern of antigen recognition shows similarity to that of iNKT
cells and MAIT cells, suggesting that : T cells:
A. Do not play an important role in immunity, but likely have a non-immune function
B. Share features of both innate and adaptive immune cells
C. Are only able to respond when the host is infected with a virus such as herpes
simplex virus
D. Are involved in maintaining the integrity of endothelial cells in the host
E. Are most important in responses to tumor cells that show stress responses

6.29 Synthesis question: A family of six (mother, father, and four children) had two children
with a history of chronic illness. Both children had repetitive infections of the sinuses,
middle ears, and lungs due to a variety of respiratory viruses. Their other siblings were
generally healthy and showed no signs of persistent or recurrent virus infections.

The two affected children had normal numbers of B cells, T cells, and NK cells in their
blood. They also showed no defects in neutrophil function or in complement protein
levels. The two children also had normal antibody levels to vaccine protein antigens,
such as tetanus toxoid, and had normal T cell responses to antigens from the vaccine
strain of Mycobacterium tuberculosis after being vaccinated.

Blood cells from one parent, one healthy child and the two affected children were
examined for surface MHC protein expression by flow cytometry using two antibodies,
one that recognizes all HLA class I proteins, and one that recognizes all HLA class II
proteins. The results are shown in Figure Q6.29A.

Figure Q6.29A

a) Analysis of HLA genotypes from the two affected children showed that they shared
one haplotype of this locus. This haplotype encodes a common HLA-A allele, HLA-A2.
Based on these data, is it likely that the two affected children have a point mutation (or
mutations) in the coding sequence for HLA-A2? Why or why not?

b) Name two proteins that could be candidates for the defective gene in the two affected
children.

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To address which gene defect might be present in the affected children, peripheral blood
cells were isolated from one healthy child and one affected child, and mRNA was
isolated from the cells. Quantitative RT-PCR was performed to assess the levels of
mRNA for the three HLA class I heavy chain genes (HLA-A, -B, and –C) and for the 2-
microglobulin gene. The results are shown in Figure Q6.29B.

Figure Q6.29B

In a second experiment, Western blots were performed, confirming that cell lysates from
both affected children contained normal levels of all three HLA class I heavy chain
proteins and the 2-microglobulin protein.

c) Do these data eliminate any of your answers to part (b)?

In a final experiment, peripheral blood cells from the two affected children and one HLA-
A2+ parent were transfected with a construct encoding the Hepatitis B virus surface
antigen (HepBsAg), a protein that is currently used in the vaccine against Hepatitis B.
This protein is normally synthesized and transported to the cell surface. In addition to the
full length HepBsAg construct, cells were also transfected with a mini-gene, encoding
just a single HLA-A2-binding peptide derived from Hepatitis B, amino acids 338–347.
Using a cytolytic CD8 T cell clone specific for HepBsAg(338–347) peptide bound to HLA-
A2, the transfected cells were tested for recognition by the CD8 T cell clone using an
assay that measures target cell killing. Figure Q6.29C shows the results of this
experiment.

Page 12 of 22
 Figure Q6.29C

d) What is the most likely gene that is defective in the affected children?

6.30 Synthesis question: In the 1980s, a mutant strain of mice was identified, carrying
amino acid changes in the MHC class II gene. This mutant strain was derived from
C57Bl/6 mice, which carry the H-2b haplotype. Inbred H-2b mice express only one MHC
class II protein, called Ab. The mutant strain, called ‘bm12’ was found to have 3 amino
acid changes in the Ab protein, at positions 67, 70, and 71 of the Aβ chain. The positions
of these amino acid changes on the MHC class II structure are shown below by the red
circles in Figure Q6.30A. On the right, the side view diagram of MHC class II shows the
direction of these three amino acid side chains.

Figure Q6.30A

Initial experiments with wild-type C57Bl/6 mice and bm12 mice showed that the wild-type
mice made a robust CD4 T cell response after immunization with the insulin protein
isolated from a cow; in contrast, the bm12 mice failed to make any detectable response
to this foreign protein. Epitope mapping studies identified amino acid residues 1–14 of
the bovine insulin A chain as the peptide recognized by CD4 T cells from wild-type mice.

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a) What is the most likely explanation for the failure of bm12 mice to make a CD4 T cell
response to bovine insulin?

In a second set of experiments, T cells from wild-type (WT) or bm12 mice were mixed in
vitro with antigen-presenting cells (APCs), in the presence or absence of the
superantigen staphylococcal enterotoxin B (SEB), and T cell proliferation was measured.
The data from these experiments are shown in Figure Q6.30B.

Figure Q6.30B

b) What is the explanation for the results in Rows 1–4 of the table?

c) Why does the T cell response to SEB (Rows 5–8) show a different pattern than the
response to bovine insulin?

d) In the table above, T cell proliferation was measured after 4 days of incubation of T
cells, APCs, +/- SEB. If one isolated the T cells at the end of the incubation for the six
conditions in which robust proliferation was seen (Rows 2, 3, 5–8), and stained the T
cells with each antibody (separately) from a panel of antibodies that recognize each of
the mouse V domains (i.e., an antibody to V1, an antibody to V2, etc), what result
would be expected?

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ANSWERS

6.1: A = ii
B=v
C = iii
Viruses and some bacteria replicate in the cytosolic compartment, as shown in panel A. Their
antigens are presented by MHC class I molecules to activate killing by cytotoxic CD8 T cells.
Other bacteria and some parasites are taken up into endosomes, usually by specialized
phagocytic cells such as macrophages, as shown in panel B. Here they are killed and degraded,
or in some cases are able to survive and proliferate within the vesicle. Their antigens are
presented by MHC class II molecules to activate cytokine production by CD4 T cells. In
response to these CD4 T cell-derived cytokines, macrophages up-regulate their mechanisms for
killing intracellular pathogens. Extracellular pathogens (or their protein productions) may bind to
cell-surface receptors and enter the vesicular system by endocytosis, illustrated in panel C for
antigens bound by the surface immunoglobulin of B cells. These antigens are presented by
MHC class II molecules to CD4 helper T cells, which can then stimulate the B cells to produce
antibody.

6.2: C.
Some pathogens may infect somatic cells but not directly infect phagocytes such as dendritic
cells. In this case, dendritic cells must acquire antigens from exogenous sources in order to
process and present antigens to T cells. For example, to eliminate a virus that infects only
epithelial cells, activation of CD8 T cells will require that dendritic cells load MHC class I
molecules with peptides derived from viral proteins taken up from virally infected cells. This
exogenous pathway of loading MHC class I molecules is called cross-presentation, and is
carried out very efficiently by some specialized types of dendritic cells.

6.3: A.
Proteins in cells are continually being degraded and replaced with newly synthesized
proteins. Much cytosolic protein degradation is carried out by a large, multicatalytic protease
complex called the proteasome. Proteins in the cytosol are tagged for degradation via the
ubiquitin–proteasome system (UPS). This begins with the attachment of a chain of several
ubiquitin molecules to the target protein, a process called ubiquitination. The general
degradative functions of the ubiquitin–proteasome system have been co-opted for antigen
presentation, so that MHC molecules have evolved to work with the peptides that the
proteasome can produce.

6.4: D.
The constitutive 1, 2, and 5 subunits of the catalytic chamber of the proteasome are
sometimes replaced by three alternative catalytic subunits that are induced by interferons.
These induced subunits are called 1i (or LMP2), 2i (or MECL-1), and 5i (or LMP7). Thus, the
proteasome can exist both as both a constitutive proteasome present in all cells and as the
immunoproteasome, which is present in cells stimulated with interferons. The replacement of
the  subunits by their interferon-inducible counterparts alters the enzymatic specificity of the
proteasome such that there is increased cleavage of polypeptides after hydrophobic residues,
and decreased cleavage after acidic residues. This produces peptides with carboxy-
terminal residues that are preferred anchor residues for binding to most MHC class I molecules
and are also the preferred structures for transport by TAP.

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6.5: Tap-1 or Tap-2.
A defect in either of these proteins would substantially reduce the availability of MHC-binding
peptides in the endoplasmic reticulum (ER), leading to poor expression of MHC class I on the
cell surface. This defect can be rectified by adding an excess of peptide to the outside of the
cell. Another possible, though less likely, candidate is ERAAP. A defect in ERAAP also affects
the peptides available in the ER for binding to MHC class I, but overall, cells do not show a
reduction in total MHC class I expression on the cell surface when ERAAP is missing. Instead,
the ERAAP defect alters the repertoire of MHC class I-bound peptides. A third, even less likely,
possibility is a defect in the proteasome. While this defect would reduce peptide availability,
cells lacking this important proteolytic machinery would not be viable.

6.6: E.
In order for MHC class I molecules to faithfully display information about the internal state of the
cell to T cells, the surface MHC class I molecules must make stable long-lasting interactions
with their peptide cargo. In other words, it is critical that very little peptide exchange occurs at
the cell surface. Should peptide exchange at the cell surface happen, infected cells would fail to
be recognized by pathogen-specific T cells; in addition, healthy uninfected cells might bind a
pathogen peptide from the extracellular milieu, and find itself a target of CD8 cytotoxic T cell
killing.

6.7: False.
In uninfected cells, peptides derived from self proteins fill the peptide-binding groove of the
mature MHC class I molecules and are carried to the cell surface. Some of the newly
synthesized MHC proteins fail to find a peptide cargo, and do undergo degradation, but this is
not the majority of MHC class I molecules synthesized.

6.8: C.
Cross-presentation is a specialized pathway found in a subset of dendritic cells. This pathway
provides a mechanism for these dendritic cells to present peptides derived from extracellular
sources on their MHC class I molecules. In other words, for these dendritic cells, the peptide
source for MHC class I presentation does not have to derive from proteins synthesized in that
dendritic cell’s cytosol. Cross-presenting dendritic cells can present peptides derived from
viruses or bacteria taken up from the extracellular milieu. This example represents an additional
mechanism for cross-presentation, where dendritic cells present peptides derived from
phagocytosed dying cells infected with a cytosolic pathogen. If the virus is inactivated, it will not
replicate in the epithelial cell, and therefore, will not generate proteins to be cross-presented by
the dendritic cell.

6.9: B.
The initial activation of CD8 T cells requires that the T cell recognizes viral peptide MHC class I
complexes on dendritic cells, not on the infected cell itself. The mechanism that the dendritic cell
uses to present viral peptides on its MHC class I proteins does not require that the virus
replicate in the dendritic cell. Instead, the virus particles can be taken up from the extracellular
milieu, or the dendritic cell can phagocytose dying infected cells. In either case, the viral
proteins will be degraded into peptides and cross-presented on MHC class I molecules. In these
scenarios, the virus is not replicating in the dendritic cell, and therefore cannot synthesize the
proteins it might use to down-regulate MHC class I in the dendritic cell.

6.10: C.
Drugs that raise the pH of endosomes inhibit the presentation of intravesicular antigens,
suggesting that acid proteases are responsible for processing internalized antigen. These

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proteases include the cysteine proteases, known as cathepsins B, D, S, and L, of which L is the
most active. Antigen processing can be mimicked to some extent by the digestion of proteins
with these enzymes in vitro at acid pH. Asparagine endopeptidase (AEP), another cysteine
protease, is important for processing some antigens, such as the tetanus toxin antigen for MHC
class II presentation. It is likely that the overall repertoire of peptides produced within the
vesicular pathway reflects the activities of the many proteases present in endosomes and
lysosomes.

6.11: False.
Trafficking of membrane proteins is controlled by cytosolic sorting tags. In this regard, Ii has a
second function, which is to target delivery of the MHC class II molecules to a low-pH
endosomal compartment where peptide loading can occur. The complex of MHC class II :
heterodimers with Ii trimers is retained for 2–4 hours in this compartment. During this time, the Ii
molecules are cleaved, ultimately leaving a single peptide (CLIP) bound to MHC class II
molecules. Peptide exchange can then occur, leading to release of CLIP and binding of
peptides derived from internalized pathogen proteins or self proteins.

6.12: A.
HLA-DM binds to and stabilizes empty MHC class II molecules and catalyzes the release of
CLIP, thus allowing the binding of other peptides to the empty MHC class II molecule. The HLA-
DM molecule does not contain the open groove found in other MHC class II molecules, and it
does not bind peptides. Instead, HLA-DM binds to the α chain of the MHC class II molecule near
the region of the floor of the peptide-binding site. This binding induces changes in the structure
of the MHC class II molecule, and holds this part of the peptide binding groove in a partially
‘open’ configuration. In this way, HLA-DM catalyzes the release of CLIP and of other unstably
bound peptides from MHC class II molecules. In the absence of HLA-DM, MHC class II
molecules assemble correctly with Ii and seem to follow the normal vesicular route, but fail to
bind peptides derived from internalized proteins and often arrive at the cell surface with the
CLIP peptide still bound. HLA-DO is as a negative regulator of HLA-DM. HLA-DO binds to HLA-
DM in the same manner as MHC class II molecules, and thereby inhibits HLA-DM from
mediating peptide editing of MHC class II.

6.13: E.
MHC class I molecules display peptides derived largely from cytosolic proteins, so it is important
that these MHC molecules do not bind to extracellular peptides. MHC class II molecules present
peptides derived from pathogens living in the vesicles, or from pathogens or antigens they have
ingested. Again, it is important that these MHC molecules do not bind to extracellular peptides.
In either case, surface MHC molecules binding to extracellular peptides could lead to healthy
cells mistakenly being targeted for destruction or activation. Fortunately, when an MHC class I
molecule at the surface of a living cell loses its peptide, its conformation changes, the 2-
microglobulin dissociates, and the  chain is internalized and rapidly degraded. Thus, most
empty MHC peptide class I molecules are quickly lost from the cell surface, largely preventing
them from acquiring peptides directly from the surrounding extracellular fluid. This helps ensure
that primed T cells target infected cells while sparing surrounding healthy cells. Empty MHC
class II molecules are also removed from the cell surface. Although at neutral pH, empty MHC
class II molecules are more stable than empty MHC class I molecules, they aggregate readily,
and internalization of such aggregates is thought to account for their removal.

6.14: B.
MARCH-1 is expressed constitutively in B cells and induced by the cytokine IL-10 in
macrophages and dendritic cells. It resides in the membrane of a recycling endosomal

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compartment, where it ubiquitinates the cytoplasmic tail of MHC class II molecules,
leading to their eventual degradation in lysosomes, thereby regulating their steady-state level of
expression. The MARCH-1 pathway is shut down during infection to increase the stability of
peptide:MHC complexes. In addition to regulating MHC class II expression in dendritic cells,
MARCH-1 also regulates expression in dendritic cells of the co-stimulatory molecule CD86 (or
B7-2), which like MHC class II molecules, is also regulated by ubiquitination. This means that by
the time dendritic cells arrive at lymph nodes, they express peptides derived from the pathogens
that activated them and have higher CD86 levels that provide signals for greater CD4 T cell
activation. Some pathogens have taken advantage of this pathway by producing MARCH-1-like
proteins to down-regulate MHC class II molecules as a means of evading adaptive immunity.

6.15: True.
When cells are treated with the interferons IFN-, -, or -, there is a marked increase in the
transcription of MHC class I -chain and 2-microglobulin genes and of the proteasome,
tapasin, and TAP genes. The increases in MHC expression these cytokines produce helps all
cells to process viral proteins and present the resulting virus-derived peptides on their surface.
On dendritic cells, this helps to activate the appropriate T cells and initiate the adaptive immune
response to the virus. In addition, gene expression of the classical MHC class II proteins, along
with the invariant-chain, DM, DM, and DO is coordinately increased by IFN-, which is
produced by activated
TH1 cells, CD8 T cells, and NK cells. This form of regulation allows dendritic cells and
macrophages to up-regulate molecules involved in processing of intravesicular antigens when
presenting antigens to T cells and NK cells. The coordinated regulation of the genes encoding
these components is likely facilitated by the linkage of many of them in the MHC.

6.16: D.
Polymorphism refers to within-species variation at a gene locus, and thus in the gene’s protein
product; the variant genes that can occupy the locus are termed alleles. For several MHC class
I and class II genes, there are more than 1000 alleles in the human population, far more than
the number of alleles for other genes found within the MHC region. This polymorphism results in
broad diversity of MHC protein expression between individuals in the population. In contrast, a
single individual may express a dozen or so different MHC proteins (MHC class I and MHC
class II), but all of the cells within the individual will express the same set of these MHC
proteins.

6.17: Polygeny and co-dominant expression of MHC proteins.

The high polymorphism of the classical MHC genes ensures diversity in MHC gene expression
in the population as a whole. However, no matter how polymorphic a gene is, no individual can
express more than two alleles at a single gene locus. Polygeny, the presence of several
different related genes with similar functions, along with the co-dominant expression of both
alleles of each MHC gene, ensures that each individual produces a number of different MHC
molecules. The combination of polymorphism, polygeny, and co-dominant expression produces
the diversity of MHC molecules seen both within an individual and in the population at large.

6.18: C.
Pathogen-driven selection can account for the large number of MHC alleles. The products of
individual MHC alleles, often known as protein isoforms, can differ from one another by up to 20
amino acids, making each variant protein quite distinct. Most of the differences are localized to
exposed surfaces of the extracellular domain furthest from the membrane, and to the peptide-
binding groove in particular. Peptides bind to MHC class I and class II molecules through the
interaction of specific anchor residues with peptide-binding pockets in the peptide-binding

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groove. Many of the polymorphisms in MHC molecules alter the amino acids that line these
pockets and thus change the pockets’ binding specificities. This in turn changes the anchor
residues of peptides that can bind to each MHC isoform.

6.19: D.
This experiment illustrates the concept of MHC restriction. When T cells are primed in a mouse
expressing H-2a, they will only recognize and kill target cells expressing H-2a MHC molecules, if
those target cells are infected with the same virus used to prime the mice. The same holds true
for mice of strain B. The fact that T cells from each mouse strain recognize their syngeneic MHC
molecules on LCMV-infected target cells rules out any lack of response of one strain to the
virus, and rules out any lack of infectivity of target cells by the virus.

6.20: E.
A child will generally share only 50% identity of MHC alleles with either parent. In this case, the
child’s T cells will respond vigorously to the non-shared 50% of peptide:MHC complexes
expressed on the parent’s antigen-presenting cells. This will lead to a robust proliferative
response by the child’s T cells.

6.21: False.
In principle, alloreactive T cells might depend on recognizing either a foreign peptide antigen or
the nonself MHC molecule to which it is bound for their reactivity against nonself MHC; these
options have been called peptide-dependent and peptide-independent allorecognition. But as
the number of individual alloreactive T-cell clones studied has increased, it seems that most
alloreactive T cells actually recognize both; that is, most individual alloreactive T-cell clones
respond to a foreign MHC molecule only when a particular peptide is bound to it. In this sense,
the structural basis of allorecognition may be quite similar to normal MHC-restricted peptide
recognition and be dependent on contacts with both peptide and MHC molecule, but in this case
a foreign MHC molecule.

6.22: A.
Superantigens are unlike other protein antigens, in that they are recognized by T cells without
being processed into peptides that are captured by MHC molecules. Indeed, fragmentation of a
superantigen destroys its biological activity, which depends on binding as an intact protein to the
outside surface of an MHC class II molecule that has already bound peptide. In addition to
binding MHC class II molecules, superantigens are able to bind the V region of many T-cell
receptors. Bacterial superantigens bind mainly to the V CDR2 loop, and, to a smaller extent, to
the V CDR1 loop and an additional loop called the hypervariable 4 or HV4 loop. Thus, the -
chain V region and the CDR3 of the  chain of the T-cell receptor have little effect on
superantigen recognition, which is determined largely by the germline-encoded V gene
segments that encode the expressed V chain. Each superantigen is specific for one or a few of
the different V gene products, of which there are 20–50 in mice and humans; a superantigen
can thus stimulate 2–20% of all T cells. This mode of stimulation does not prime an adaptive
immune response specific for the pathogen. Instead, it causes a massive production of
cytokines by CD4 T cells, the predominant responding population of T cells. These cytokines
have two effects on the host: systemic toxicity and suppression of the adaptive
immune response. Both these effects contribute to microbial pathogenicity.

6.23: C.
These arguments raise a question: if having three MHC class I loci is better than having one,
why are there not far more? The probable explanation is that each time a distinct MHC molecule

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is added to the repertoire, all T cells that can respond to self peptides bound to that MHC
molecule must be removed to maintain self tolerance. It seems that the number of MHC genes
present in humans and mice is about optimal to balance the advantages of presenting an
increased range of foreign peptides with the disadvantages of losing T cells from the repertoire.

6.24: True.
In addition to the highly polymorphic ‘classical’ MHC class I and class II genes, there are many
‘nonclassical’ MHC genes encoded in the MHC (as well as others encoded outside this region).
The MHC class I-type molecules show comparatively little polymorphism; many of these have
yet to be assigned a function. These genes have been termed MHC class Ib genes. Their
expression on cells is variable, both in the amount present at the cell surface and in tissue
distribution. The functions of some MHC class Ib proteins are unrelated to the adaptive immune
response, but many have a role in innate immunity by interacting with receptors on NK cells.
The other genes that map within the MHC include some that encode complement components
(for example, C2, C4, and factor B) and some that encode cytokines—for example, tumor
necrosis factor- (TNF-) and lymphotoxin—all of which have important functions in immunity.
Finally, some genes residing within the MHC have no known or suspected immunological
function.

6.25: C.
Members of the MIC gene family are MHC class Ib genes that are expressed under a different
regulatory control than the classical MHC class I genes and are induced in response to cellular
stress (such as heat shock). The MICA and MICB proteins are recognized by the NKG2D
receptor expressed by NK cells. In addition, NKG2D is also expressed by : T cells and some
CD8 T cells, and it can activate these cells to kill MIC-expressing targets. NKG2D is an
‘activating’ member of the NKG2 family of NK-cell receptors; its cytoplasmic domain lacks the
inhibitory sequence motif found in other members of this family, which act as inhibitory
receptors. Even more distantly related to MHC class I genes is a small family of proteins known
in humans as the UL16-binding proteins (ULBPs) or the RAET1 proteins. These proteins also
bind NKG2D. They also seem to be expressed under conditions of cellular stress, such as when
cells are infected with pathogens or have undergone transformation to tumor cells. By
expressing ULBPs, stressed or infected cells can bind and activate NKG2D molecules
expressed on NK cells, : T cells, and CD8 cytotoxic : T cells, and so be recognized and
eliminated.

6.26: A.
The T cells that recognize lipids presented by CD1 molecules are largely negative for CD4 and
CD8 expression, although some express CD4. CD1d-restricted T cells have a T-cell receptor
repertoire that is substantially less diverse than conventional : T cells. In fact, the majority of
these cells use the same TCR chain (V 24–J 18 in humans). In addition, these cells also
express NK-cell receptors. These CD1-restricted T cells are called invariant NKT (iNKT) cells.
The ability of iNKT cells to recognize different glycolipid constituents from microorganisms
presented by CD1d molecules places them in an ‘innate’ category, while their possession of a
fully rearranged T-cell receptor, despite its relatively limited repertoire, makes them ‘adaptive’.

6.27: Microbial products specific to folate metabolism.

Analysis of MR1 proteins that were refolded in the presence of supernatants from cultures of
Salmonella typhimurium eventually led to the identification of several riboflavin metabolites that
are formed by biosynthetic pathways in most bacteria and yeast. These metabolites not only
bind to MR1, but also activate MAIT cells. Thus, MAIT cells are activated in response to

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infection by these organisms by detecting products specific to their folate metabolism. As such,
MAIT cells appear to hold an intermediate place in the spectrum of innate and adaptive
immunity, similar to iNKT cells, in that they use an antigen receptor assembled by somatic gene
rearrangement, but recognize a molecular structure that falls within the definition of a PAMP.

6.28: B.
The function of : T cells has remained somewhat obscure, due primarily to difficulty in
identifying the ligands they recognize. Yet the abundance of : T cells across vertebrate
species, their rapid expansion to form more than 50% of the blood lymphocytes during
infections, and their abundant cytokine production all argue for an important role in immunity.
Over time, many different ligands recognized by : T-cell clones have been identified, and their
diversity suggests that, like iNKT and MAIT cells, they hold an intermediate, or transitional,
position in the spectrum of innate versus adaptive immunity.

6.29:
a) No. The antibody used to detect MHC class I protein expression on the surface of cells
will recognize all MHC class I proteins, including HLA-A, HLA-B, and HLA-C proteins.
Since the affected children show no MHC class I expression at all, their cells are unable
to express any of the genes. Therefore, the defect cannot be in just the gene encoding
HLA-A. This is likely a genetically inherited immunodeficiency disease, with each parent
being heterozygous for gene defect. It is unlikely that a single HLA locus would have
inactivating mutations in all three HLA class I genes. Instead, it is more likely that the
affected children inherited a deficiency in a single gene that is encoded outside of the
HLA locus; furthermore, this gene must encode a protein that is required for all surface
HLA class I protein expression.
b) β2-microglobulin or TAP-1 (or TAP-2). Two proteins required for MHC class I surface
expression are 2-microglobulin and a component of the TAP complex (TAP-1 or TAP-
2). In the absence of 2-microglobulin, HLA class I heavy chain proteins will not fold
properly, will not bind peptide, and will not traffic to the cell surface. In the absence of the
TAP complex, there will be an insufficient supply of peptides in the endoplasmic
reticulum. In this case, again, HLA class I proteins will not fold properly and will not traffic
to the cell surface.
c) Yes. These data rule out a defect in the 2-microglobulin gene, as both the mRNA and
protein for 2-microglobulin are expressed normally in the cells from the affected
children.
d) TAP-1 or TAP-2. The data from the cytolysis experiment show that the parent’s cells will
present the HepBsAg 338–347 peptide on HLA-A2 when they are transfected with either
the full-length HepBsAg gene or the mini-gene encoding just the peptide epitope. In
contrast, the cells from the affected children will only present this peptide from the full-
length construct, not from the mini-gene. This indicates that the cells are able to present
the HepBsAg epitope when the protein containing that epitope is able to get into the
endoplasmic reticulum (ER). Since all surface proteins have signal sequences that target
them for translocation into the ER, the full-length HepBsAg protein will access this
mechanism. Once in the ER, the normal proteolytic enzymes will degrade the protein,
and peptides will bind normally to HLA-A2 during synthesis and assembly. The mini-
gene encodes only the 338–347 amino acids of the HepBsAg, and therefore lacks any
signal sequence for translocation into the ER. This peptide will be synthesized and
remain in the cytosol. These data localize the defect in the affected children’s cells to the
mechanism that normally mediates transfer of cytosolic peptides into the ER, i.e., the
TAP complex.

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Test Bank for Janeway’s Immunobiology 9th Edition

6.30:
a) The bovine insulin A (1–14) peptide most likely fails to bind to bm12. The amino acid
changes in bm12 compared to wild-type Ab will alter the peptide binding specificity of this
MHC class II molecule. The lack of CD4 T cell response to other regions of the bovine
insulin protein in bm12 mice is due to the fact that the majority of sequences comprising
the bovine insulin protein are conserved between mouse and cow insulin. The amino-
terminal region of the insulin A chain is the major area of divergence between the insulin
proteins from these two species.
b) The T cells from WT mice recognize peptide:MHC complexes on bm12 antigen
presenting cells as ‘foreign,’ and vice versa for the T cells from bm12 mice. This is due
to the fact that the WT Ab molecules bind a distinct set of peptides compared to the
bm12 Ab molecules. Even though the T-cell receptors on the T cells do not directly
contact the altered amino acid residues, they ‘see’ this amino acid variation due to the
broad effect of these substitutions on the global repertoire of peptides bound to WT Ab
versus bm12 Ab.
c) The T cell response to SEB occurs due to SEB binding to the side of the MHC class II
protein. Since the amino acid differences between WT and bm12 Ab lie inside the
peptide binding groove, they are not exposed to the surfaces that directly contact the
SEB or the T-cell receptor. Therefore, T cells from each strain of mice will recognize
SEB when bound to APCs from either strain of mice.
d) The proliferating T cells in Rows 2 and 3 would be heterogeneous. All (or nearly all) of
the different Vs would be detected in the population, but at a variety of different
frequencies. Some would be more abundant, and others very rare in the overall
population. In contrast, the T cells in Rows 5–8 would all show predominant expansion
of cells with one single V domain. In the case of C57Bl/6 mice and SEB, this is V8. In
some cases T cells expressing one of two different Vs may be expanded. The key point
is that all four populations should look the same, and show nearly homogeneous
expansion of T cells whose T-cell receptors use a single type of V domain.

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