Immunology Review: GOOD LUCK ON THE FINAL EXAM!

Our major focus is the adaptive immune system – types of cells that produce receptors (either
antibodies or T-cell receptors) that will be able to recognize either parts of proteins, carbohydrates,
lipids (in the case of Abs) or parts of proteins (in the case of T-cell receptors).

The innate immune system is important as well – the main important type of cell we are interested in for
this system is the macrophage. This type of cell will eat or phagocytose “foreign-looking things” in its
path non-specifically.

B cells are capable of producing a diverse set of antibodies. Yet a single mature B cell is capable of
producing only a single antibody. How is this the case?

An antibody is the product of two individual proteins formed into a complex. These proteins are termed
the heavy chain and light chain. When two of each of these come into a complex, an antibody is
formed.
We know that this antibody will be able to specifically recognize
something about molecules. How does this work? In order to
understand this, you must remember the protein-protein interaction
material that we began the course with. We looked at several amino
acids, that is, the building blocks of proteins, and we decided that some
amino acids like to be in close proximity. They are happy being next to
each other. For example, a positively charged amino acid like lysine
will be happy being near a negatively-charged amino acid like aspartate,
rather than even a neutral amino acid like leucine. So, we can think
about a given molecule, perhaps part of a protein that may enter into our
systems… if we desire to recognize this protein as foreign, we must design an antibody (that is, design a
protein) that will be compatible to these sequences. Our bodies generate millions of unique antibodies in
hopes of recognizing a variety of future invaders. If our antibody is compatible and binds these
sequences, we will get binding and an immune response.

How do we make all of these different antibodies? This is accomplished by gene-rearrangement or

VDJ recombination. If we begin with a B cell progenitor, this cell will not yet be making any
antibodies. However, it will have a heavy chain gene and a light chain gene that are not yet being
transcribed. Something must happen in order to allow for the transcription and translation (production of
Ab) to occur. We will choose one of each of these segments to actually be a part of the eventual heavy
and light chain genes that are transcribed – one V region of 100, one D region of thirty and one J region
of 6.

This allows for a very large number of possible heavy chain genes that we can make. Once we have
selected these gene segments and the light chain gene segments (in an analogous process), we will
actually begin to make antibodies, by first transcribing the heavy and light chain DNA and translating the
mRNA that is made. Once this antibody is made, a decision will be made about whether we are interested
in having the antibody in our system. That decision revolves around whether the antibody recognizes
something that is already in our systems – something that is a “self antigen”. If this is the case, the B-
cell will be signaled to die and the antibody will thus cease production. This is called clonal deletion or
negative selection. If not, the B-cell precursor will go about searching our bodies for foreign antigens.

A similar process occurs for T cells. We know that we have two different populations of T cells, but each
begins at the same point in development with a T cell precursor. This precursor cell expresses both the
CD4 and CD8 receptors that are associated with helper T cells and cytotoxic T cells respectively. These
T cells will make a gene rearrangement of the alpha and beta genes that code for proteins of the T-cell
receptor.

These rearrangements will generate diversity in that the resulting T-cell

receptors will recognize different peptides depending on what sequence
they have included. Again, there is a negative selection process in the
production of T cells. If a T-cell recognizes “self antigen” during the
maturation process, it will be signaled to die. Once the T-cell receptor
gene rearrangements have been made, there will be a further
differentiation of T cells into helper T cell (CD4) and cytotoxic T cell
(CD8) populations. We will now review the roles of these different
populations.

Let’s begin with a crucial event that

happens in our systems. A B cell
which we have produced with a
unique antibody on its surface
recognizes a foreign antigen in our
systems. What is the result? Well,
this foreign antigen could be any of the pathogens we have talked
about – it could be freely floating bacteria or a virus that has not yet
infected a cell. (Remember during this entire section that this B cell
could be swapped for a macrophage which will engulf antigens non-
specifically but process them in a similar fashion). The recognition of
a foreign antigen by a B cell will be accompanied by an important
event – the internalization of the complex of antibody and antigen.
This internalization will allow the B cell (or again, macrophage) to
begin degrading the bacterial or viral proteins to much smaller
peptides. These peptides will be placed on the surfaces of the B cells
using special receptor complexes called MHC class II or major
histocompatibility complex II.

This is the B cell sending out a warning signal to your system. It has
found something foreign and now it needs help to respond appropriately to the intruder. This help will
come in the form of a helper T cell. The helper T cell with a proper T-cell receptor and CD4 receptor on
its surface will recognize the B cell with the MHC II receptor “presenting” the antigen. This formation
of a complex between the two cells allows for a very
important event called clonal expansion. The helper
T cell will signal the B cell to proliferate, thus
making many more B cells producing the same
antibody. Additionally, you will produce cells that
secrete this SAME antibody (plasma cells) and
memory cells that will be set aside to respond to the
same antigen in the future. With the new infection,
you would be able to generate many more antibodies
and much more quickly. And secreted antibodies are capable of coating free-floating antigens, triggering
their engulfment.
Also, the helper T cell will undergo clonal expansion, thus making many more T cells that will respond
this the same peptide (or protein fragment) sequence recognized by this helper T cell.

While extremely useful, none of the antibodies we have

discussed have the ability to enter cells of your body. For this
reason we have another type of immunity – cellular immunity. It
is called this because it is dependent upon another cell (a “host”
cell) to generate an immune response. All cells of our body are
able to play a role in this process. When a cell is infected, it will
go through a series of events to signal cells of your immune
system that something is wrong. Once a virus enters a cell and
begins to replicate … or a bacterium enters a cell, the proteins of
that virus or bacterium will be floating around in your cell.
Fortunately, we have vesicles called lysosomes in our
cells filled with enzymes that will break down these proteins.
These vesicles do this to all of your proteins – not just viral and
bacterial proteins – such that you can get rid of old ones. Once
the proteins are broken down into peptides, these peptides
will be “presented” on the surface of cells on another MHC
receptor (this time, MHC class I). And once again, these
peptides placed on the surface of cells will be recognized by T
cells (this time, cytotoxic T cells).

A cytotoxic T cell with a unique T-cell

receptor that recognizes the peptides of
your virus or bacterium will find the
MHC I receptor and form a complex,
along with the CD8 receptor on its
surface. Here, we have a similar
complex as above for the B cell and
helper T cell, but this time we will have a
different response. Last time, we wanted to expand the populations of each of these immune cells types
to find more antigens in our system. This time, our prime goal is to eliminate one of these cells – the host
cell. This is accomplished by having the cytotoxic T cell release perforin (which will poke holes in the
infected cell) and granzyme (which will signal the infected cell to apoptose, or undergo programmed
cell death). Additionally, the cytotoxic T cell will undergo clonal expansion, or proliferate, such that it
may find and kill other infected cells in our system.

Through each of these responses (humoral – B cell or macrophage – antibody – helper T cell) (cellular –
normal infected host cell – cytotoxic T cell), we generate unique outputs that are best for dealing with
each type of antigen. If we merely had a system to take care of free-floating antigens, we would be
helpless against viruses that had already infected cells, and vice versa.

Prion Review:

When we talked about the immune system, we discussed several types of pathogens that were composed
of genetic material and perhaps proteins surrounded with a lipid and/or protein coat. In fact, this is such a
standard way of thinking about infectious agents that another type of infectious agent, prions, took a long
time for people to accept.

When we were discussing the origins of cancer research, we talked about how the introduction of an
activated oncogen into a viral genome made that virus “transforming”. It was able to “transform” normal
cells into cells that had lost control over their proliferation. However, in this case, this process of
transformation included the introduction of foreign DNA into a cell. This DNA was translated into
protein and the protein caused the cells to “misbehave”.
In thinking about prions, we are dealing with rogue proteins as well. However, there is no requirement
for the introduction of DNA sequences encoding mutant proteins. Rather, the infection takes place
entirely at the protein level. This is because a certain form of the protein is able to “poison” the rest of the
population of protein.

In this case, we are talking about the PrP protein that is

commonly found in our cells. Normally it takes on its own
form and performs its own function. However, once
another form of the protein is introduced into the cell, the
normal protein goes bad. What can happen is that the
normal protein will adopt an identical conformation as the
bad protein and these proteins together will form
aggregates or clumps in cells. These aggregates are bad
news for the cells and can lead to their demise.

Prions have been associated now with diseases in sheep, cattle, and humans and the major worry is that
the transmission is interspecific. That is, scrapie, the disease in sheep was able to cause “mad cow
disease” (or BSE, bovine spongiform encephalopathy) in cattle, which was able to cause Creutzfeld-
Jakob disease in humans. Though the PrP encoded by each of these species’ genomes is likely
somewhat different at the sequence level, the “scrapie” version of each protein is able to have ill-effects
on the “normal” version in another species.

While we have talked about our immune systems as being potentially successful in dealing with foreign
antigens because they look different. Or perhaps using special drugs to fight the ill-effects of HIV, these
types of things are completely ineffective against Prion diseases. In fact, the only thing that has been
shown to be successful in destroying the protein (even keeping the protein in formalin for many years did
not destroy it) is by using chemicals that will unfold proteins. That is, unfolding the scrapie
conformation of the protein. And clearly, this is not a viable treatment for human beings with Creutzfeld-
Jakob disease.
Cancer

Definition: uncontrolled cell proliferation

Principle: Remember the cell cycle? Mitosis? A cell will only
divide (go through the cell cycle) when specific signals in the
environment tell it to do so, for example, during development,
for normal tissue turn-over, or in some cases for regeneration of
damaged tissue. Growth Factors (GF) are the substances in
the environment that tell cells to divide. Genes regulating the
cell cycle are mutated in cancer.
Two main types of genes regulate progression through the cell
cycle:
• (Proto-)Oncogenes: Signal the cell to proceed through
the cell cycle (step on the gas pedal) in response to
growth signals
• Tumor Suppressor Genes: Signal the cell to stop (step
on the brake) in the absence of growth signals, presence of inhibitory signals or wrong
conditions such as DNA damage
These proteins act at certain points of the cell cycle and tell the cell to proceed if division signals
are present or to stop if these signals are absent.

How does this lead to cancer?

The regulation by these proteins is

very tightly controlled. If this control
is disrupted by mutation of these
proteins, the cell will be signaled to
divide when it shouldn’t and that’s
how we get uncontrolled cell
proliferation. If a mutation happens
in a:
• Proto-oncogene (transforming it into an oncogene) the cell will be signaled to divide
without need for growth factors. One hyperactive copy is enough to make the cell
proceed through the cell cycle. We refer to this type of mutation as a gain of function
mutation because it makes the protein be active (stepping on the gas pedal) all the time.
At the cellular level, this mutation acts as dominant.
Examples: Ras G-protein unable to hydrolyze GTP  GDP to turn off.
Constitutively active growth factor receptor (no need for GF)
Bcl-2: usually inhibits apoptosis. If mutated, it will inhibit apoptosis all
the time (cells won’t die when they should)
• Tumor suppressor genes: If mutated, it can’t stop progression through the cell cycle. If
only one copy is mutated, the second one is enough to regulate. We need a mutation in
BOTH copies for loss of regulation. This type of mutation is called loss of function
mutation because the protein function is lost. At a cellular level, it acts as recessive
(but at the organism level it looks like dominant! Pedigrees). When a heterozygote
individual looses the healthy copy we say that there was Loss of Heterozygosity (very
common, this is why it appears as dominant on pedigrees of familial cancers).
 Examples: pRb (retinoblastoma): acts at
restriction point. In presence of
GF, it gets phosphorylated,
becoming inactive to allow cell
cycle progression. When not
phosphorylated (active) it prevents
cell cycle progression.
p53: usually promotes apoptosis in
response to damage. If mutated, it
can’t signal cells to die.

Types of mutations that lead to oncogenic transformation:

1. Point mutation: overactive Ras,

tyrosine kinase GF receptor
2. DNA amplification: many copies
of a gene (GF Receptor)
3. Translocation: chromosomes
break and attach to other
chromosomes. Usually messes up
regulation. Can generate protein
fusions. Examples:
a. Translocation causes gene
to end up in front of
strong promoter. Gene
will be transcribed all the
time. (Example: myc)
b. Protein fusion. Example:
BCR-ABL. Kinase is not
regulated anymore.
Phosphorylates substrates ad libitum. Phosphorylated substrates signal cell to
divide.
4. Viral integration (retroviruses):
a. Virus can carry an oncogene (Rous sarcoma virus)
b. Virus can carry a strong promoter and disrupt regulation of a gene (would cause a
gene to be constitutively expressed.

Cancer associated mutations affect:

a. Proliferation: cause increase

b. Cell death (apoptosis): cause decrease
c. Angiogenesis: Increase in cells’ ability to recruit blood supply to tumor mass to get
nutrients
d. Cell motility: This is the basis for metastasis (cancer travels to other tissues). Mutations
that increase cells’ ability to move, degrade extracellular matrix, enter blood vessels,
travel and invade other tissues contribute to increased metastatic potential (cancer is more
aggressive).
e. Invasion: Cells have an increased ability to establish a tumor in a different tissue
Remember that as cancer proceeds, the cells acquire more mutations that make the cancer more
aggressive.

Cancer treatment

The way that proteins communicate with each other usually involves a change or conformation,
or phosphorylation (or both!) Let’s consider the case of BCR-ABL. BCR-ABL is a kinase (it
phosphorylates a specific substrate). This implies that at least two sites must exist on the enzyme:
1. ATP-binding pocket
2. Active site (substrate-binding site)

Gleevec, a drug developed against BCR-ABL binds tightly to the ATP-binding pocket thus
preventing ATP from binding there. Therefore, the substrate can’t be phosphorylated. This is a
strategy to circumvent the overactive phosphorylation by BCR-ABL.
However, in many cases a relapse is observed in patients treated with Gleevec. This usually
occurs because BCR-ABL acquires a new mutation that changes the conformation of its ATP-
binding pocket. Gleevec cannot bind tightly anymore and is less effective. We say that BCR-
ABL is now resistant to Gleevec.
Viruses

Viruses are obligate parasites. This means that they can only replicate once they have infected a
host cell (the replication and/or transcription and translation machinery of the infected cell is
exploited by the virus in most cases).
Viral genomes evolve very rapidly, which means that they acquire multiple mutations with
every generation.

Virus components

• Genome: RNA or DNA/ single

stranded (ss) or double stranded
(ds)/ fragmented or in a single piece
• Capsid: Protein coat that protects
viral genes and allows viral genes
to gain entrance to host cells.
Capsid proteins are ALWAYS encoded by the viral genome. (Capsid + genome =
nucleocapsid)
• Lipid bilayer: only present in enveloped viruses. Usually from host cell origin (as the
virus is budding off of the host cell, it takes some membrane with it).
• Viral encoded capsid proteins: these proteins are studded on the membrane surrounding
the nucleocapsid. They allow binding to a specific receptor on the host cell.
• Enzyme needed to replicate genome: (only some viruses MUST carry these proteins with
them at the time of infection, for example, reverse transcriptase for retroviruses. Others
can translate them when they infect the cell and thus they don’t need to carry a physical
copy of the protein).

Steps in viral infection

1. Adsorption (attachment): Virus binds a specific cell surface proteins to gain entry to a
specific type of host cell
2. Penetration can happen by endocytosis (by attaching to a surface protein on a piece of
membrane that is internalized) or by direct fusion of viral and host membranes (only for
enveloped viruses).
3. Genome replication can happen in the cytoplasm of the host cell or in the nucleus
depending on the viral replication strategy
4. Host cell take over. Virus causes a decrease in host cell mRNA translation (forces host
cell to focus exclusively on translation of viral proteins) and an increase in cell cycle
activity (to promote replication of viral particles).
5. Viral assembly: once they have been made using the cell’s resources, the viral
components are assembled
6. Viral release can happen by cell lysis (the cell “explodes” and releases viral particles.
Usually for naked viruses) or by budding (for enveloped viruses)

Double-stranded DNA viruses

Genome goes to the nucleus where it acts as an extra chromosome (mimics host cell DNA).
Uses host DNA polymerase (for replication), RNA polymerase (for transcription of viral
genes), ribosomes (for translation of viral mRNA).
What if a virus infects a cell in Go? This cell will be quiescent, so it won’t be replicating its
DNA. To circumvent these problems, SOME dsDNA viruses may carry their own DNA
polymerase or a protein that makes the host cell enter the cell cycle (this division-promoting
protein can function as an oncogene to make the infected cell cancerous). In addition to these
proteins (if present) the viral genome usually encodes structural genes such as the capsid
proteins.
Examples: herpes virus family, Epstein-Barr virus (mononucleosis), human papilloma virus or
HPV (cervical cancer), adenovirus.

Single-stranded DNA viruses

These viruses usually have small, simple genomes. First, the virus needs to make dsDNA to
serve as a template for transcription (ssDNA is not a template for RNA polymerase!). The virus
uses the host’s DNA polymerase to make the complementary DNA strand. But, wait a
minute; doesn’t DNA polymerase need a primer? Yes! These smart viruses use the 3’ end of
their DNA as a primer, as shown in the drawing to the left.
3’ This way a ds version of the viral genome is made, which
5’ serves as a template for transcription. Those transcripts are
used to make viral proteins. The viral DNA is converted back
into a ssDNA genome and the virus is packaged for export.
Examples: canine and feline parvo viruses

Single-stranded RNA (+) viruses

Single stranded RNA (+) genome is formally and functionally identical to mRNA. After
penetration, this viral genome is translated directly by the host cell to make viral proteins.
These viral proteins include an RNA dependent RNA polymerase that uses the RNA (+) as a
template to make complementary RNA (-) that are then used as templates by this same enzyme
to make new ssRNA (+). These newly synthesized ssRNA (+) molecules are destined to serve as
genome or as mRNA to produce more viral proteins. The ssRNA viral genome usually codes for
several proteins but is initially translated as a large polyprotein that is then cut into the
separate viral proteins by a viral protease.
Examples: common cold virus, poliovirus.

Single-stranded RNA (-) viruses

Their genome is complementary to the necessary viral
mRNA so it can’t be directly translated.

Because its genome cannot be translated directly after

penetration and because the host cell does not have an
enzyme that can make the (+) RNA strand using the (-)
strand as a template, these viruses MUST carry their
own viral RNA dependent RNA polymerase in their
nucleocapsid. This enzyme can make full length RNA
(+) copies to be used as templates for making full
length genome copies. The enzyme can also make
shorter RNA (+) used for translation of viral proteins.
Some ssRNA (-) viruses have segmented genomes, so
packaging must be regulated to ensure that the
progeny viruses receive one copy of each segment.
Examples: measles, influenza, rabies, Ebola, vesicular stomatitis virus (VSV)
Double-stranded RNA viruses
These viruses are naked and they have a fragmented genome. They carry an RNA dependent
RNA polymerase to translate the dsRNA into ssRNA (+) that can then serve as mRNA for
translation of viral proteins or as a template for (-) strand synthesis and formation of the dsRNA
genome.

ssRNA (+) Retroviruses

These are enveloped viruses. Their replication strategy is very

different from that of ssRNA (+) viruses such as poliovirus.
After entering the cell, the RNA (+) does not serve as an
mRNA, instead it is used to make a DNA copy of the viral
genome. The host cell also lacks an enzyme that can make
DNA from RNA, so the virus carries an enzyme in its
nucleocapsid called Reverse Transcriptase (RT), an RNA
dependent DNA polymerase.
RT first makes a DNA copy of the RNA(+). Next, the RNA is
degraded and RT synthesizes the complementary strand of the
newly made DNA strand to obtain dsDNA. This dsDNA is
transported to the nucleus where it is integrated and
covalently linked to the host chromosomal DNA by the viral
enzyme integrase (provirus = viral DNA integrated into the
host genome).
Provirus is used for transcription of viral mRNAs to make viral
proteins and as template for new genomes for viral progeny.
Example: HIV
HIV binds to the CD4 protein on the membrane of helper T cells to gain entrance into the cell.
After an initial response where the immune system clears most part of the virus, the virus that
had become integrated and had remained latent in some cells (so those infected cells were not
killed) can be stimulated to reproduce. When the latent virus reproduces, viral proteins are
expressed on the infected cells’ MHC I, so they are cleared by cytotoxic T cells. Eventually, the
T helper cell population is depleted and virus concentration increases. The humoral immune
system is paralyzed without T helper cells (B cells can’t become activated, antibodies can’t be
produced). Finally death occurs by opportunistic infections.