Antigens

The technical definition of antigen is anything that can be used by the immune system to provoke a response. Our bodies are bombarded constantly by foreign substances and pathogens. It is our Immune Systems job to sort out the friendly from the harmful. It is designed to tolerate friendly bacteria and target and destroy harmful pathogens and substances. Most of the time, it gets it right, but sometimes it makes mistakes. These mistakes can lead to allergies and autoimmune disorders. Understanding antigens and how the immune system handles them is the most important concept of immunology.

So what is an antigen? It is a small piece of a pathogen or substance that immune cells can pick up and recognize to mount an immune response. The key is antigens are not the whole pathogen. It is just a part of that pathogen or substance. They are made up of the four basic building blocks of life with Nucleic Acids, Proteins, Lipids and Carbohydrates. Even toxins can be an antigen like heavy metals, snake venom and poison ivy. They can even be innocuous substances like pollen or pet dander. In some cases, even our own proteins, lipids, carbohydrates and nucleic acids can become antigen for an immune response. This can occur when the immune system targets cancer cells or in autoimmunity.

One key concept of antigens is surface bound vs free floating. Some antigens are bound to the surface of a cell or a pathogen like a bacteria or virus. Other antigens are free floating in the blood or fluids like bacteria, pollen or snake venom. The immune system will respond to different types of antigens differently so it is important to understand the difference between surface bound antigens vs those that can float freely. When it comes to T cells and B cells, they don't use the same types of antigens. The B cells are designed to find those cell surface and free floating antigens in circulation. The T cells are designed to find damaged or infected cells.

T cells are solely focused on proteins and pieces of proteins. The B cells can respond to anything including proteins, carbohydrates, lipids, nucleic acids, poisons, toxins and even unharmful things like pollen or dust. There are a set of immune cells called Antigen Presenting cells (APCs). They line all the tissues which are in contact with the outside world like the Respiratory tract, GI tract and skin. Their job is to pick up anything foreign that comes into the body and process it. These tend to be cells like Dendritic cells, B cells and Macrophages, but many other cells can set off the alarm like Mast Cells. The job of the antigen presenting cell is to ingest and break down pathogens and present their pieces to T cells.

The T cells will check the antigens presented by the APC and determine if and what type of response is needed. The immune system is trained to tolerate self or friendly antigens while responding to non self or harmful antigens. When Immunology goes wrong, it happens somewhere in the APC processing antigens, presenting antigens or the T cells responding incorrectly. Allergies stem from APCs presenting unharmful substances as if they were pathogens by mistake.

The term Epitope is also important to understand in immunology. The epitope is a specific piece of a larger protein that the immune cells can respond to. Proteins can be very big and can break down into thousands of pieces. Any of those pieces can become an epitope. The T cell receptor binds to a sequence of amino acids of 7 to 10 amino acids long while the B cell binds to a sequence around 25 amino acids long. The actual protein can be hundreds or even thousands of amino acids long. That means two different antibodies or T cells could target different parts (epitopes) of the same protein. It is a key concept to understand in immunology as it affects how two drugs can have dramatically different effects.

The last concept for antigens to understand is the Neoantigen. This is an important concept in oncology. It happens when a protein mutates. That mutation makes the protein that was otherwise friendly and tolerated by the immune cells is now different. These unique antigens created by a protein mutation will allow the immune system to potentially target the cancer cells with that new neoantigen and kill them. These unique mutations can be antigens to target with antibodies or T cells. There has been a lot of science around using these neoantigens to develop personalized cancer treatments designed for each patient. The importance of neoantigen is that each mutation is unique to each cell of each tumor of each patient. It is very rare that two proteins in two different patients mutate in the exact same way.

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Organs of the Immune System

The Immune System is unique compared to other systems of the body. That is because immune cells need to move around and respond anywhere in the body to reach sites of infection. This mainly includes the Lymphatic System, the Circulation of the Blood, and the Tissue. The organs of the immune system are organized into the Primary and Secondary organs. The primary organs are where the immune cells are created and trained. The secondary organs are where the immune cells live and work. The two primary organs of the immune system are the Bone Marrow and the Thymus. The bone marrow is where most cells for the immune system are produced.

In the Bone Marrow all cells come from the Hematopoietic stem cells. The immune cells are the Leukocytes which means white cells. We often refer to them as the White Blood Cells (WBCs). There are about 10 immune cells made in the bone marrow. Each one of these cells has a different role. The Leukocytes are broken down into 3 groups. The fist is the Granulocytes. They make up the Neutrophils, Basophils and Eosinophils. Their role is around fighting Bacteria and Parasites. The next group of Leukocytes are the Monocytes. They circulate in the blood for a while before they migrate into the tissue where they mature into the Macrophages and Dendritic cells that are Sentry cells. The last group of Leukocytes is the Lymphocytes. The Lymphocytes make up the NK cells of the innate system, and the T cells and B cells of the Adaptive Immune System. Every cell of the immune system comes from the Blood with rare exceptions like tissue resident Macrophages.

This makes the Bone Marrow the key Primary organ for the immune system. The other primary organ is the Thymus. This is an organ in the chest where T cells go for training. This is where T cells are trained for self tolerance. Once all the immune cells are made, they will move into circulation. They will travel through the blood going from lymph node to lymph node patrolling the circulation. Most immune cells will not leave circulation into the tissue unless they are called there by inflammation. The secondary immune organs are those of the circulation like Lymph Nodes, Tonsils and the Spleen. The cells of the immune system, especially those of adaptive immunity like T cells and B cells, will spend their time circulating between lymph nodes.

When inflammation occurs, the cells of the immune system will respond and traffic through the blood to the site of infection where they will travel out of the blood and into the tissue. They will fight the infection. Some smaller elements of the blood will naturally move into the tissue like water, antibodies and complement proteins. All of the fluids that are in the tissue will then drain into the lymph system and back to the lymph nodes where it is screened by the immune cells for pathogens. That makes the process where it pushes out of the blood, into the tissue, drains into the lymph system, gets screened, and it is finally returned to the blood circulation.

So how does this whole thing work? The Sentry Cells will sit in the tissue where they look for pathogens. When they find one, they ingest it and break it down. They leave the tissue by the lymph drainage which takes them to the nearest lymph node. Through the lymph nodes, you have constant circulation of T cells and B cells. The Sentry cell will present the antigens to those T and B cells to activate them. The activated T and B cells will expand into an army. This is why lymph nodes swell and get painful. That army of T and B cells will leave the lymph node into the blood circulation. They will see the inflammatory signals in the circulation at the site of the infection. That will guide them out of the blood and into the tissue where they will attack the pathogen. As the infection is cleared, all the extra immune cells will die off and the immune system will return to its resting state. The only thing left behind will be a few memory immune cells to stand guard should we encounter that pathogen again.

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Innate vs Adaptive Immunity

The immune system is broken down into 2 parts. The first is the innate immune system and the second is the adaptive immune system. The innate immune system is made up of Physical and Chemical barriers along with the Sentry cells. The adaptive immune system is made up of the T cells and B cells. There are some differences that define each of these systems.

The first is the innate system is always active while the adaptive takes about 5 to 10 days to start working. The innate immune system is made up of many physical barriers for pathogen entry. The skin is the largest of the immune organs. It prevents infections from entering the body. Maintaining good integrity of the skin is important to stop infections. The next part of the innate system is chemical barriers. We produce a lot of proteins and enzymes that are present in our fluids like the tears and saliva that are destructive to some pathogens. This helps reduce the level of exposure. Even the pH acidity of the stomach does its part to wipe out many pathogens. The last part of the innate system is the sentry cells in the tissue. All tissue that makes contact with the outside is lined with sentry cells.This makes the innate immune system always active. It never takes a day off, and it works day and night.

On the other side is the adaptive immune system. It is made up of T cells and B cells. They reside in the lymph nodes in the inactive state. They get activated by the sentry cells. They take days replicating in the lymph node before there are enough to respond. This can take between 5 and 10 days. This is why we often feel sicker for the first week or so with a cold or flu. Then the adaptive immune system kicks in and we start to feel better over time.

The next difference between innate and adaptive immunity is how specific they are in their response. The physical and chemical barriers of the innate system are just there. It only keeps out what it is designed to keep out. The sentry cells can recognize pathogens, but only in a limited fashion. They can determine what kind of pathogen they are fighting like a virus or bacteria. They can not tell one type of bacteria from another. They use pattern recognition receptors that help them find specific patterns in pathogens that stand out from us. These are things like double stranded RNA. Human cells do not have double stranded RNA. That is an immediate danger signal to sentry cells. The adaptive cells with T cells and B cells, can be extremely specific. Not only will they know what kind of pathogen they are facing like virus or bacteria, they will know exactly what specific virus they are facing like influenza or rhinovirus. The adaptive T and B cells can be so specific and diverse that they can make many different responses to the same pathogen. The typical flu virus could generate many T cells and B cells all attacking different antigens of the pathogen.

The last major difference between the innate and adaptive immunity is memory. The innate system returns to the same state after each infection. It approaches every infection in the same way. It approaches every infection from starting over. The adaptive immune system has memory. When T cells and B cells are created toward a pathogen, some of them will become long lived memory cells. They can live for years or even decades. This gives us protection from previous exposure to a pathogen. Memory for a pathogen can still be elusive. Some pathogens, especially viruses, can mutate rapidly and completely change their antigens. The flu is one such virus that remains elusive as it mutates dramatically. This constant battle between immunity and pathogen mutations can be endless with viruses like the flu. It is still proven that protective immunity through antibodies from memory cells can significantly improve outcomes in infections.

The flu virus has about 5 antigens that the immune system can make antibodies toward. They rapidly mutate to change to avoid the immune system. If you even have 1 antibody that knows just 1 of those antigens, it is shown that it can reduce the severity of infection by 20% or more. This is why vaccination becomes very powerful and important. It gives the immune system memory without the need for infection thus conveying protection. For pathogens that never mutate, this can be life long protection. Smallpox was eradicated due to vaccination. That was a very deadly and horrible virus. Other viruses that mutate frequently, like the flu, are a constant battle, but having some antibodies can make a huge difference.

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Physical and Chemical Barriers

The innate immune system is made up of three barriers that try to prevent pathogens from getting into our bodies. They are the physical barriers, chemicals barriers and the sentry cells. The first is the Physical barrier of the skin. It covers our body and prevents pathogens from entering the body. The outer layer of skin is covered with dead skin cells that shed to remove pathogens. Many infections will enter the body through a break in the skin. The skin also contains defensins which are small proteins that can puncture a pathogen and damage it. The skin contains a huge amount of friendly bacteria that live on it. This friendly bacteria, called Flora, will occupy space preventing bad pathogens from getting in. The skin is the largest single part of the immune system. It represents the largest single barrier to infection. The number 1 cause of death in burns is infection. That is due to the loss of this key barrier to infection.

Then there are the mucous linings that coat the sinus, throat, GI tract, lungs and even reproductive tracts. This mucus is slimy and helps to keep the pathogens from getting a foothold. Sometimes the flow of mucus will carry away pathogens before they can gain infection. Most of the mucus contains enzymes like Lysozyme which can break down the walls of bacteria. Some of the other physical and chemical barriers of the innate immune system are the low Ph of the stomach. This can break down many pathogens. Only the strongest encapsulated viruses can survive the low pH of the stomach. Even the reproductive tract has a lower pH to help try to thwart pathogens which try to enter. There are small hair like structures that line the lungs called cilia. These cilia of the respiratory tract can move pathogens out of the lungs to help protect it. This is what causes coughing to help expel foreign particles and pathogens.

The last major defense of our bodies from pathogens is friendly bacteria called Flora. All of the linings of our skin, upper respiratory tract and entire GI tract is lined with friendly bacteria. The lower respiratory tract is the exception. These friendly bacteria simply occupy space that prevents other more harmful bacteria from occupying. These bacteria not only help us with blocking bad bacteria, but they have functions like helping digestion. Antibiotics will wipe out friendly bacteria in the GI tract. Probiotics will help replenish the GI tract with friendly bacteria so harmful ones can not move in and colonize the GI tract. Many bacteria in the bacterial world are in a fight of survival. They will often release substances that will kill off other nearby competing bacteria. Some of these substances are what cause our illness toward these bacteria. Others we have harnessed as natural antibiotics toward harmful bacteria.

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Sentry Cells

There are several cells that make up the cells of the innate immune system. They typically share a few similar attributes. Innate cells play a sentry role being located near entry points where pathogens enter the body. The innate cells are always lying in wait for a pathogen to come along. They will be the first cells to respond to a pathogen.

First comes the Basophil and Mast cell. They are the same cells with the same role, but the Basophil is in the circulation. When it moves into the tissue, it will differentiate into a Mast Cell. They have granules inside their cytoplasm that is released when they come in contact with pathogens. They have several receptors for pathogen detection and can respond to signals released from other cells or complement proteins. They play a major role in allergic reactions and anaphylaxis. They release histamine which is a massive inflammatory mediator that leads to vasodilation, increased vessel permeability and bronchoconstriction.

Next up is the Eosinophil which plays a major role in parasite infections. These will bind to the parasite and release their granules. They are made up of granules in their cytoplasm. They contain Major Basic Protein which can break down the parasite's surface. This will puncture the parasite and cause it to die.

Then comes the Neutrophil. This is one of the most important of all the innate cells. It is the most abundant, making up 70% of all the white blood cells in our bodies. They play a big role in fighting bacterial infections. They make up puss with that yellowish green tint that comes from the Myeloperoxidase they use to destroy bacteria. They are one of the key cells in inflammation as they rush into the area to fight infections. They can digest (phagocytose) bacteria, break it down and spit it back out. The neutrophil is called the professional phagocyte for this reason. They also contain granules which can be released. These granules contain enzymes that can damage pathogens and friendly cells alike. This plays a role in tissue damage during infections and inflammation. They can also deploy Neutrophil Extracellular Traps (NETs). When a bacteria infection gets too intense, the Neutrophil will spit out its DNA like a web catching up as much bacteria as it can. The neutrophil will digest about 20 bacteria before it dies. The neutrophil has a nucleus that is made up of multiple lobes usually in the 3 to 5 lobes range. This has earned them the name polymorphonuclear (PMN) cells. This can be diagnostic as megaloblastic anemias will have hyper segmented Neutrophils of more than 5 lobes. The neutrophil uses enzymes to digest pathogens, but some pathogens will be too strong for this process. The neutrophil can use Radial Oxygen Species (ROS) which can blast these pathogens with oxygen radicals to destroy them. This is an important process to understand as neutrophils can cause a ton of tissue damage from oxidative stress. If you ever heard the term antioxidants, that is what they do. They help reduce the oxygen radicals caused by tissue damage. The ROS process takes Oxygen + NADPH oxidase enzymes to create O2++ radicals. Then it takes Superoxide Dismutase (SOD) to turn that into hydrogen peroxide (H202). This will kill most pathogens. When necessary, they can use myeloperoxidase to create hypochlorite (CLO-) bleach. Only neutrophils have this myeloperoxidase enzyme to take their ROS abilities to that next level of destroyer. This also gives them their yellow/greenish color which is why puss/phlegm can be yellow or green. Neutrophils will ingest, destroy and spilt out pathogens like a killing machine. They typically kill about 20 pathogens before they die themselves. They will then become pus or phlegm and eventually get cleaned up by the Macrophages.

Now we will start to look at the Monocytes which only have a single nucleus and do not have any granules. They start as the Monocytes in the blood which circulate. They either get called into the tissue by inflammation or do so with age. They circulate for about a day before leaving into the tissue where they will differentiate into Dendritic Cells and Macrophages. These are different from the tissue resident Macrophages.

The Dendritic Cells are the second phagocyte and the first Antigen Presenting Cell (APC). They are cells with very big tentacles that make them look like an octopus. They get their name from the dendrites of the nervous system as they look similar, but they have a completely different role. They use a system called Pinocytosis which means they drink in and sample everything around them. They act like giant filters for pathogens. When they encounter a pathogen they will mature. They leave the tissue where they live and head for the nearest lymph node to present their antigens. This process has earned them the name the professional APC. The lymph nodes have their own version of the Dendritic Cell called the Follicular Dendritic Cell (FDC). They come from different origins and present pathogens in the lymph nodes to T and B cells. They also participate with B cells for antigen maturation.

The Macrophage is the most important cell in all innate immunity. They are both phagocyte and antigen presenting cells. They can act as sentry cells sitting in the tissue looking for pathogens. They can also act as generals in the tissue directing the battle against infections. They have the ability to switch roles from killer to clean up. They fall into two roles of the M1 and M2 macrophages. The M1 macrophage is in the battle mode for infections. They can phagocytose pathogens and release all kinds of inflammatory signals to call in all the other immune cells to the source of the infection. The macrophage uses pattern recognition receptors to find pathogens. It can break them down and present those antigens to the adaptive immune cells. It will release proinflammatory cytokines like TNF-a, IL1-b, CXCL8, IL-6 and GM-CSF. These signals will play a role in the immune response like inflammation to recruit other immune cells and growth factors to stimulate the growth of more neutrophils and macrophages. The M1 macrophage will have some of the same enzymes as the neutrophil. They have the ability to make Radical Oxygen Species (ROS) like the neutrophil, but only up to the hydrogen peroxide stage. They do not have myeloperoxidase. When the battle is over, they will switch to M2 mode and begin cleaning up. They will ingest and break down dead neutrophils and cells. They will secret signals to promote cell repair and stop the immune response. They will release cytokines like IL-10 and TGF-beta that stop the immune response and activate the repair systems. The M2 macrophages are cleaner and builder. Some tumors will exploit this by releasing cytokines that cause macrophages to switch from M1 which would kill the tumor to M2 which will not be able to respond to the tumor. Macrophages also have antibody receptors to work with antibodies of the immune system to clear pathogens. They also work with complement proteins to help clear infections. The macrophage is one of the only 3 immune cells capable of killing other cells. Most cells are way too big to be handled by immune cells. Only the T cells, NK cells and Macrophage are capable of killing a cell. The macrophage can engulf and destroy pathogens up to a certain point for antigen presentation, but for full activation, it needs to get proper signals from Helper T cells to become fully activated. The macrophage comes in two types based on their origin with resident and infiltrating. The tissue resident macrophages all have fancy names like Langerhans in the skin, microglia of the brain or alveolar macrophages in the lungs.

The last innate cell is the Natural Killer cell. This is a very close cousin to the T cell. Their role is to look for any cells that look damaged or defective. They use several unique receptors called Damage Associated Molecular Patterns (DAMP) receptors. They use these DAMP receptors to look for cells that might be distressed, damaged or infected. NK cells have CD16 receptors and work with antibodies in a process called Antibody Dependent Cellular Cytotoxicity (ADCC). This allows them to bind to cells or pathogens identified by antibodies. The NK cell will measure the level of MHC expressed on cells to ensure they are healthy. They have other receptors that will look for other signs of stress from cells like Mic-A or Mic-B. They play a key role in initial detection of viral infections and cancer surveillance. The NK cell is the only innate cell that does not come from the myeloid blood lineage. It comes from the lymphocytes along with T cells and B cells. Unlike other lymphocytes, NK cells spend most of their time in the tissue instead of the lymph nodes.

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PAMPS

We hear about the receptors of immune cells every day in biotech like T cell receptors or B cell receptors, but we never hear about the many receptors of the innate immune cells. The innate cells use receptors called Pattern Recognition Receptors (PRR). They look for very specific patterns that pathogens have that our bodies do not have like flagellin, lipopolysaccharides, and double stranded RNA. There are many of these receptors, but one kind you must know for anyone studying immunology is the Toll Like Receptors (TLR). These are used by all the innate cells to look for these Pathogen Associated Molecular Patterns (PAMPs). The Toll Like Receptors (TLR) look like a question mark. They can look for Lipopolysaccharides (LPS) of gram negative bacteria, or they can look for Lipoteichoic Acid (LTA) of gram positive bacteria. Or many many other patterns. They can look for double strand RNA or even single stranded DNA. These receptors look for patterns that are not natural to our body. This becomes a warning sign that a pathogen is present. These receptors are used by key innate cells like the Antigen Presenting Cells (APCs).

There are a total of 9 TLR receptors that we know what they do for sure. Not every innate cell will have all 9 of these PRRs, but they will have the ones necessary for their role. These receptors allow an innate immune cell to determine if a pathogen is a bacteria, a parasite, or a virus and react appropriately. Some of these receptors are on the surface of the cell while others are inside the cytoplasm. Some of the TLRs are on the cell surface like TLR 1, 2, 4, 5, and 6. Then there are some that are inside the cell like TLR 3, 7, 8, and 9. This is designed for types of pathogens that get inside the cell. Most bacteria stay outside the cells and live in the tissue. The receptors to detect them are on the outside surface of these innate immune cells. A virus works by invading a cell and taking it over. The receptors for detecting them are inside the innate immune cells.

The toll-like receptors work together as pairs called a dimer. When they bind to a pattern, they come together in a set (dimer) and activate. They don't always have to be the same TLR either. Two of the same would be called a homodimer. The TLR1 works with TLR2, and TLR2 can also work with TLR1 or TLR 6. The rest work with the same kind as TLR4 works with TLR4 and so on. When 2 different TLRs come together to form a dimer, it is called a heterodimer.

The TLR 4 is used for gram negative bacteria. This is used by mast cells, neutrophils and macrophages. This is probably one of the most common and active TLRs used by many innate cells. The role of the toll-like receptor is to detect a pathogen and activate these innate cells so they can alert the rest of the immune system that an invader has been detected. The Mast Cell uses TLR 4 or other signals to degranulate and release histamine which is a proinflammatory signal which will call other immune cells to the scene. The dendritic cells and macrophages are the other primary sentry cells that can use most of these pattern recognition receptors to find all kinds of pathogens. They will release signals called cytokines to alarm other cells like neutrophils to come and join the fight.

Regardless of which TLR becomes activated by the pathogen, there are just 2 responses that will happen. The first will be the release of Interferons for the "antiviral state" and the other will be the inflammation response with TNF-a and IL-1b. Understanding Toll like receptors is critical to infections, cancer responses and autoimmunity with inflammation. Most autoimmune disorders and allergies are linked to inappropriate responses by TLR to innocuous substances.

There are some other types of pattern recognition receptors like RIG and NOD receptors. There are also Lipopolysaccharide and Mannose receptors on innate cells. The innate immune cells use these PAMPs to find and detect pathogens and alarm the rest of the immune system. They are not designed for a great level of specificity. They can only find basic patterns. There is no way they can tell one bacteria from another or one virus from another. Their job is to start the inflammation process and present these antigens to the adaptive immune cells. Many autoimmune disorders can be attributed to innate cells picking up self antigens as if they were pathogens. This has a lot to do with different genetics across the genes that make up these PAMP receptors. It is a very large area of study.

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Complement

Complement is a group of proteins that are created by the liver and circulate in the bloodstream. It is designed to complement the rest of the immune system with fighting infections. There are over a dozen proteins that play a role in the complement system. We cover the key 9 that starts at C1 and goes to C9, but there are many others that play a role in the process as inhibitors and regulators of complement. The complement proteins are produced in the liver and circulate in the blood. They can pass into the tissues. The complement proteins circulate in their inactive form. They get activated by pathogens or other immune proteins. Once activated, they act like a cascade where 1 protein activates 1 or more other proteins which combine to make and active enzyme complexes which activate other complement proteins.

The complement system is made up of 3 pathways. They all start at different places, but they all end up at the same result. The 3 pathways of complement are the Alternative, the Lectin and the Classical Pathways. The Lectin and Alternative fall under the innate immune system as they are capable of triggering on their own in response to pathogens. The Classical pathway is part of the adaptive immune system as they work with antibodies once they are produced.

The purpose of the complement is 3 fold with inflammation, opsonization and lysis. Complement causes inflammation to alert other immune cells that an infection exists. It coats or opsonizes the pathogens to make them easier for sentry cells to engulf and break them down. The final role of complement is lysis. This is the process of creating small holes in the membranes of pathogens to kill them.

There are 2 key complement proteins in this system. The first in the C3 protein which is responsible for causing opsonization of pathogens with C3 complement proteins. This makes it easy for sentry cells to bind to the C3 with their C3 receptors and engulf and destroy the pathogens. When the C3 is activated, it gets broken into 2 pieces called the a and b fragments. The b fragment is what coats the pathogens. The a fragment creates inflammation to call in the sentry cells like neutrophils into the space to kill the pathogens. Many sentry cells will have complement receptors for binding C3. That makes C3 a critical protein for the complement system as it drives the process of opsonization. All the complement pathways start at different places, but they all reach and activate C3 to get the common process of opsonization going. The C3 protein on the surface of the pathogen will recruit another complement protein called Factor B. This binds to C3 and then gets cleaved and activated by Factor D. The activated Factor B will combine with the C3 and create an enzyme complex called the C3 Convertase. This can activate hundreds of more C3 than just having C3 alone on the pathogen surface. This is a key process of complement to reach C3 activate Factor B to form that C3 convertase and drive tons of C3 activation and opsonization of the pathogens. It becomes a chain reaction that continues to amplify as it goes.

The other key complement protein is C5. Once the C3 convertase is formed on the pathogen surface, it will also begin to recruit, cleave and activate C5. The C5 protein breaks into 2 pieces which are the a and b fragments. The b fragment will bind to the pathogen surface and start another chain reaction. The a fragment will cause inflammation and call in more sentry cells to kill pathogens. Once the C5b is on the pathogen surface, it will create a C5 convertase by binding with the C3 convertase. The C5 convertase will recruit C6, C7 and C8. These three proteins form a complex that looks like a drilling rig. Then the C9 is used to drill through the membrane of the pathogen. Many of the C9 proteins will be used. This creates a hole in the membrane of the pathogen. When this happens in mass, it creates many holes in the pathogen which causes it to fall apart. This is called Lysis. The C5 convertase on the surface of the pathogen drives the process of forming these holes in the pathogen surface. The complex it builds is called the Membrane Attack Complex using C6, C7, C8 and C9. This is known as the MAC complex. The whole process is to drive the rupture of pathogens and their death. This is a critical process of the innate system to control pathogens until the adaptive immune system can kick in. It is also a source of many immune disorders that stem from deficiencies or overactivity of the complement system. Auto Immune responses of complement against Red Blood Cells or Platelets leads to destruction of these cells and immune disorders.

Sometimes you will hear this process called the Final Common Pathway of complement. It includes the process of complement where C3 coats pathogens and activates C5 which then creates a MAC and kills the pathogen. That is because this process is common to all 3 of the complement pathways. Now we will look at the 3 pathways of complement and where they start. We will see how they reach the point of where they activate C3 and the Common Pathway.

The Alternative pathway is the easiest to cover. It starts with Lipopolysaccharides on the surface of a bacteria. This alone will trigger the C3 and begin to deposit the C3b fragments on the pathogen surface. Then Factor B will come in and bind to the C3b and be activated by Factor D. This creates the C3bBb complex on the pathogen. This is called the C3 Convertase. The C3 convertase will go on to activate large amounts of C3 and deposit the C3b fragments on the pathogen surface causing opsonization. The C3a fragment will go on to cause inflammation. The C5 will come in and bind to some of the C3 convertases and be activated. This will create a new C5 convertase with C3bBbC5b. This goes on to recruit the MAC complex proteins and drive lysis. There is a safety mechanism on this Alternative pathway. Since the C3 can activate spontaneously, there are 2 inhibitors that circulate in the blood called Factor H and Factor I. These factors are designed to bind to any activated C3 that is floating free in the blood. This prevents them from landing on our cells and triggering complement against our cells.

The Lectin pathway of complement starts with a protein that circulates in the blood. It is called Mannose Binding Lectin. This protein is made in the liver and circulates in the blood. It binds to Mannose. The Mannose is a special sugar structure that is found on some bacteria that is not on our healthy cells. The Mannose Binding Lectin (MBL) will bind to this mannose and begin the Lectin Pathway. The MBL has 2 active serine complexes on it called the Mannose Associated Serine Protease or MASP1 and MASP2. These 2 enzymes will recruit and cleave both C4 and C2 of complement. These will be broken into 2 fragments with an a fragment and a b fragment. The C4b and C2a fragments will bind to that pathogen and create a C3 convertase complex of C4bC2a. That will go and recruit and activate C3. Now we are already up to the final common pathway as C3 will go on to do what it does and opsonize. When C3 is activated, some of it will bind with the C4bC2a and form another complex of C4bC2aC3b which is another version of the C5 convertase.

The last pathway of complement is part of the Adaptive Immune system. It is called the Classical pathway. It mostly starts with Antibodies that bind to pathogens. The Fc region of the antibody has a place where the C1 complement protein can bind. It has serine proteases called C1r and C1s. They will recruit and activate C4 and C2. The Classical Pathway works exactly like the lectin pathway except it starts with the C1 complement protein instead of MBL. There is one way the Classical Pathway participates in the innate immune response. There is a protein produced by the Liver during the acute phase of Inflammation called C Reactive Protein or CRP. This protein is designed to bind to Phosphocholine. This can be expressed by stressed or damaged cells. The CRP will bind to the Phosphocholine and recruit C1 and trigger complement activation through the Classical Pathway. There is also an inhibitor in this pathway called C1 esterase. The liver produces a small amount of C1 esterase which binds to and blocks active C1. This works as a safety. When small amounts of C1 are activated, it will be blocked by C1 esterase. When antibodies are present and binding complement, there will be high levels of activation that will overwhelm the C1 esterase. This C1 esterase acts like a buffer so that only high levels of activation are taken seriously. Any low level of C1 activation will be inhibited.

There are a few other safeties on the Complement System which are important to know. These are Decay Accelerating Factor (DAF) also called CD55. The other is MAC Inhibitor Protein also called CD59. All cells in the human body that have a nucleus will make these 2 key proteins. The CD55 will block and remove any active C4 or C3 on a healthy cell membrane. The CD59 is designed to remove any MAC complexes as they try to form on healthy cells. These are 2 very important protective systems in cell biology and immunology. There are diseases that stem from the loss of these key proteins. The other issue is those cells that don't have a nucleus. This is common for Red Blood Cells and Platelets which don't have these CD55 and CD59 proteins. They often get hit by complement in autoimmune disorders.

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Adaptive Immunity

When it comes to the adaptive immune system, there are two types of responses. Pathogens will either enter cells and hide inside them, or the pathogens will stay outside the cells and live in the tissue and fluids. Some pathogens do both by replicating inside a cell then passing through the tissue and fluids to the next cell. The immune system uses two different responses to target each type of these pathogens. The first response is called the Cell Mediated response. When a cell is infected or mutated with cancer, the solution is to kill that cell before it can spread. This is done by a few immune cells that are capable of cell killing like T cells, NK cells and Macrophages. The second response is called the Humoral response. This uses many of the immune system cells and proteins to target pathogens in the fluids or tissues. It is handled by complement and antibodies of the B cells.

What determines which response is used? This is done by the Helper T cells. They act like the directors of the immune system. Most of the time, both of these responses will be active against pathogens like viruses. The Sentry Cells like Dendritic Cells and Macrophages will take the antigens they find during the innate immune response by presenting antigens to the Helper T cells to activate the Adaptive Immune response.

The Cell Mediated response can be activated by Antigen Presenting cells when they present antigens to the T cells. This can be done by processing antigens they engulf from infected or mutated cells. They will process these antigens and present them to Helper T cells and Cytotoxic T cells. They will release cytokines like IL-12 that will guide the helper T cell toward a Cell Mediated response. The helper T cells will activate the Cytotoxic T cells which will seek out and kill any infected cells.

Activation of the Humoral response starts when Antigen Presenting Cells present the antigens in the presence of IL-4 and IL-5. This will be antigens they processed from engulfing pathogens in the fluids or tissue. This causes the T helper cells to activate B cells which will produce antibodies. The antibodies can circulate the body and into the tissues. They can bind to pathogens and neutralize them until other immune cells can deal with them. Antibodies will trigger complement, NK cells and Macrophages.

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Antigen Processing and Presentation

When it comes to presenting MHC I class antigens, all nucleated cells can do this. These antigens come from foreign things that get into the cells like a virus, micro bacteria or even a cancer antigen. Inside every cell, within the cytoplasm, is a specialized cell machine called the proteasome. This machine is the recycling bin for proteins. The cells make proteins all day long. When those proteins are no longer needed, they get tagged for recycling by this proteasome. The Proteasome chops up these proteins in peptides of about 7 to 10 amino acids in length. These small amino acid peptides will be in the cytoplasm, but the MHC I proteins are in the Endoplasmic Reticulum (ER). They need to get together for the MHC I to bind the peptides. This is done by a small active transporter on the surface of the ER called Transporter Associated with Antigen Processing (TAP). This pumps these peptides into the ER. Once inside the ER, the MHC I will bind to any peptide that matches its receptor. Once it has picked up a matching peptide, it will carry it to the cell surface and display it. These peptides in the presence of MHC I will be presented to T cells to prime them for activation. Most of the time these are self proteins that are presented to T cells. The T cells know these friendly self proteins and do not react. When they are not self proteins presented, the T cells will react.

This class I presentation can be done by any cell with a nucleus, but only the Antigen presenting cells can travel to the lymph nodes where the T cells hang out to present to the helper T cells. The priming of T cells takes multiple steps that act as a safeties to ensure that the T cell response is not activated unless it is necessary. The first step is the MHC I presented by the APC which includes the foreign peptide that will bind to a matching T cell receptor for the Helper T cell. This will activate one pathway in the T cell through the T cell receptor complex. The second signal comes from the antigen presenting cell. The activated Antigen Presenting Cell will express ligands on its surface called B7-1 and B7-2 or CD80 and CD86. This will engage with the CD28 receptors of the T cell adding a second signal of co-stimulation. The third signal of activation will be the cytokines (cell signals) secreted by the APC like IL-12 in the case of an infected cell. This will tell the Helper T cell it needs a cell mediated response. The cytokines help direct the helper T cell to what response is correct.

This process of antigen presentation by the APC can happen at the same time with Helper and Cytotoxic T cells in the process of cross presentation. This is where the APC shows both cells the antigen and the helper T cell activation helps activate the Cytotoxic T cells. Antigen Presenting Cells have both MHC I and MHC II so they can present antigens to both Helper T cells and Cytotoxic T cells at the same time. The whole process of Antigen Presentation is the link between the Innate system with Sentry Cells and the Adaptive immune system with the T cells and B cells. This is the priming of the adaptive immune response by the innate cells. This whole process can take days. This is why the adaptive immune response is much slower than the innate immune response. Once active, the Helper T cells will release new cytokines that will help the innate cells like Macrophages to increase pathogen killing abilities.

MHC II is about the handling of the immune response to things outside of the cells. This is the humoral response. It can be in the blood, fluids or tissues. This can start with a pathogen like a virus or bacteria. It can also be an allergen, toxin or even a foreign protein. The Sentry cells are guarding every entrance of our body from pathogens. Some of these cells are also Antigen Presenting Cells (APC). That means they can engulf these substances like a pathogen or toxin and present them to the Helper T cells. There are 3 antigen presenting cells in the immune system with the Dendritic cell, the Macrophage, and the B cell. They all have pattern recognition receptors (PRR) to detect pathogens like a bacteria. We looked at the pattern receptors like the Toll Like Receptors. The technical name for the process of an APC ingesting a pathogen is called Phagocytosis and these cells are called phagocytes. They will engulf the pathogen into a bubble (phagosome) and take into their cytoplasm.

Inside the APC, the phagosome will merge with a lysosome of the cell which has all kinds of enzymes that will break down the pathogen into tiny pieces. These pieces will become antigens. The MHC II is made inside the Endoplasmic Reticulum just like the MHC I. The first thing you might wonder is how does the MHC II not bind to the MHC I peptides already in the ER? This is prevented by the Class II invariant chain peptide (CLIP). When the MHC II is created, it will be bound with this CLIP peptide to block its receptor so it can not bind any Class I peptides in the ER. When the CLIP reaches the phagosome where all the dead and broken up pathogens are, the CLIP will be degraded by the same enzymes that helped break down the pathogen. This releases the CLIP. Once the CLIP is released, the MHC II can bind parts of the pathogen before being trafficked to the cell surface for presentation.

Once the APC has processed the antigen and displayed it on its surface, it will now be active. It will leave the tissue and head to the nearest lymph node to present these antigens to helper T cells. This will prime the T cells and initiate the adaptive immune response. The process of presentation will undergo the same 3 steps as we saw in MHC I presentation. It will bind the antigen to the T cell receptor, it will co-stimulate with CD80 and CD86, and it will release cytokines to help the T cell move to humoral response. Depending on the antigen presented, It can activate either the Cell mediated response with cytotoxic T cells or it can activate the humoral response with B cells. Or both.

B cells are both antigen presenting cells and effector cells as they become active and create antibodies. They can bind many kinds of proteins, lipids, carbohydrates, and nucleic acids. They can take what they find and present it directly to helper T cells, and the Helper T cell can return the favor by activating the B cell. The humoral response is carried out by the helper T cell then activating B cells to become plasma cells that produce antibodies. In most infections both MHC I and MHC II presentation will happen. Both Cell Mediated and Humoral responses will be activated. T cell participation in the response is critical for B cells to undergo affinity maturation and class switching which allows them to become better at fighting a specific antigen.

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Recombination

Both T cells and B cells undergo the process of genetic recombination. Their receptors are generated by randomly selecting genes from a very broad range of possible genes. This random genetic selection process is so similar between both T cells and B cells that it makes sense to cover them together. The key to remember is recombination happens at the DNA level, and it is permanent. When it comes to selecting genes, each cell must choose randomly from three different classes of genes with Variable, Diversity and Junctional classes. This is where the recombination process gets its name as VDJ recombination.

Each Variable gene will have many possible genes to choose from just as each Diversity and Junctional will have several genes to pick from. The number of genes available to choose from depends whether it is a T cell or B cell doing the picking. The process of recombination actually occurs with the DNA. It can only occur with one chromosome. The cell must pick the mom chromosome or the dad chromosome. This is called allelic exclusion.

The T cell receptors have two chains with the Alpha chain and the Beta chain. The Beta chains will choose a Variable from 52 possible gene choices, then a Diversity from 2 possible gene choices, and finally a Junctional from 13 possible gene choices. The Alpha chain only chooses from 70 Variable gene choices and then from 62 Junctional gene choices. This allows every T cell to have a completely unique receptor for recognizing a wide variety of antigens.

The B cell receptors have two chains to choose from with the Heavy chain and the Light chains. There are two different light chains for the B cells with kappa and lambda light chains. Both these light chains use the same genes with 70 Variable gene choices and 5 Junctional gene choices. They just do it from different chromosomes. The kappa light chain draws its genes from Chromosome 2 while the lambda light chain draws its genes from chromosome 22. The Heavy chain of the B cell will pick from 40 possible Variable gene choices, 23 Diversity gene choices and 6 Junctional gene choices. This will give each and every B cell a completely unique receptor. When all three VDJ genes come together, they create three special regions of the receptor called Complementarity Determining Regions (CDR) regions. The Variable gene makes up the CDR1 and CDR2 regions while the Diversity (when used) and Junctional make up the CDR3 region. The area of the CDR3 is the most variable region of the entire receptor being called the hypervariable region. This applies to both the T cell and B cell receptors.

The process of recombination is regulated by a few elements. The first is called the Recombination Signaling Sequences (RSS). These are special sequences at the beginning and end of every gene from the Variable, Diversity and Junctional genes. The RSS sequences ensure the genes get spliced together in the right way. Each has a set of heptamer and nonamer sequences that are like pointers to show the right direction. The 12 base and 23 base segments ensure that the V, D and J segments get put together in the right order. This is called the 12/23 rule for recombination.

Then comes the Recombination Activating Genes (RAG). The RAG enzymes will randomly land on one of the RSS sequences for the Variable and Diversity genes. Then they fold the DNA and cut it. They will take one Variable gene and paste it to one Diversity gene discarding all the DNA in between. Everything that is cut out is rolled up and discarded. This leads to the cell having just one of the Variable and Diversity genes. Then the RAG proteins will land on a random Junctional gene and cut and paste that to the Variable and Diversity it previously selected to the chosen Junctional gene. There are several enzymes that come together to paste the DNA back together after it is cut by the RAG enzymes. The key one is Terminal Deoxynucleotidyl Transferase (TDT) which is important to spicing the two pieces of DNA back together. When this process occurs, an enzyme will clean up the ends of the DNA by removing some nucleotides. Then another enzyme will randomly add extra nucleotides between the two genes splicing them back together. This further adds more diversity to each and every T cell or B cell receptor that is created. The random insertion of a few nucleotides in that junctional splicing makes every T cell and B cell receptor unique, building a very diverse population.

The VDJ recombination process is designed to use enzymes like RAG to take 1 copy of each gene from Variable, Diversity and Joining and paste them together to form the T cell or B cell receptor. This means some segments of DNA will be cut out and discarded while others will not need to be. If the Variable gene 39 of 40 is chosen, then only the 40th variable segment is removed and 1-38 still remain. This is a key concept to remember as B cells can undergo recombination multiple times using the remaining gene segments if they fail to get a good receptor the first time. T cells do not have this option and undergo cell death if they fail to get a good receptor. Recombination is how each and every T can B cell creates a receptor that is unique to that cell. This gives us diversity in our immune system so we can respond to as many pathogens as possible.

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