Immunology Review: GOOD LUCK ON THE FINAL EXAM!web.mit.edu/7.01x/7.013/pdfs/sp2006/freview.pdf · Immunology Review: GOOD LUCK ON THE FINAL EXAM! Our major focus is the adaptive immune

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. This way a ds version of the viral genome is made, which 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)

5’ 3’

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.