Introduction to HLA

I am going to layer these topics for Human Leukocyte Antigens on top of each other with the first being a general high level overview. Then I will continue to stack on top of that getting deeper and deeper into the concepts of HLA.

So what is this Human Leukocyte Antigen (HLA) anyway? It is a basic friend or foe identifier for our cells. Each cell gets stamped with a set of proteins that marks that cell as ours. I love to think of it as a barcode. If you have ever gone through the self checkout line at Walmart, you scan the barcode on each item. When you do, the computer knows that exact item you scanned was Smucker's Grape Jelly for $4.49. In the same way, our bodies stamp every cell we produce with a barcode of proteins that label it as ours. Each person will have a different barcode with a different set of proteins. Every cell in the body with a few exceptions gets stamped with this HLA barcode. So why is this important? This is used by our immune systems to determine what is friendly and what is foreign. The immune cells are trained to ignore cells with the proper HLA codes. The immune system will attempt to attack and kill anything that lacks an HLA code or has the wrong HLA codes. This helps the immune system identify pathogens which want to invade us and use us to make more pathogens.

In exchange, many pathogens learn to subvert the HLA of the cells by blocking its expression so they can hijack a cell and turn it into a pathogen factory. It is a constant battle between the immune system and pathogens. The other place this HLA barcode comes into play is in transplants. You probably see this all the time on TV doctor shows. They match 2 patients to see if they can be a donor. This is exactly how it sounds. They will test people to see if their HLA barcode matches the HLA of the person who needs the organ. The genes that encode these proteins are some of the most polymorphic in the human genome. There are potentially tens of thousands of combinations of the genes that make these HLA proteins. To be a match, they have to match every protein perfectly. There are 6 proteins on every cell that have to be matched. When the HLA does not match you have rejection. This comes in two kinds with Graft vs Host and Host vs Graft. This might sound complicated, but it is not once I explain it.

We stated the immune cells use the HLA to determine which cells are friendly or foreign and it tries to kill anything foreign. If you put immune cells from one person into another, they would not recognize that person's HLA and thus attack all their healthy cells. This is Graft vs Host. The transplanted cells are attacking the recipient. This is the case in stem cell transplants since stem cells produce the immune cells. In Host vs Graft rejection, the person who receives the cells will have their immune system target and kill those cells because the donor organ does not have the correct matching HLA.

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HLA Genes

The HLA genes are located on the short arm of chromosome 6. They are a group of genes. We will focus on 6 of them, but there are many more. Some of them we will cover in cell therapies. The first concept we must address is that of a haplotype. A haplotype is a group of genes that are inherited together. They get inherited together from each parent. You get 1 of your mom's number 6 chromosomes and the exact copies of her HLA genes from that chromosome. You get 1 copy of your father's number 6 chromosomes and the exact copies of his HLA genes from that chromosome. Barring any mutations, you get the exact same copy of these genes from a single parent chromosome. These genes are broken into 3 classes, of which, we will only focus on HLA class I and HLA class II genes.

This is where I got to introduce another term called Major Histocompatibility Complex (MHC). What in the world is that? It is an immunology term used to describe the proteins created by the Human Leukocyte Antigen (HLA) genes. When we are speaking of the genes or the alleles of those genes, we use the HLA. When we are referring to the proteins made from those genes that get used by the immune system, we call them MHC. The HLA genes will be divided into 2 classes with HLA Class I and HLA class II. These genes create the MHC proteins which are named the MHC Class I and MHC Class II proteins. So remember HLA = the genes and MHC = the proteins made by those genes.

There are 3 genes that fall under HLA class I titled HLA-A, HLA-B and HLA-C. No points for creativity there. These 3 genes encode 3 different proteins of Class I MHC. There are potentially thousands of alleles for each of these HLA-A, HLA-B and HLA-C genes. Since you get 2 chromosomes 6's, with one from mom and one from dad, you end up with 6 total HLA Class I genes. That means every cell has 2 each of HLA-A, HLA-B and HLA-C proteins. Most likely they are all from different alleles with one copy of each coming from each parent. In the best case scenario, we would want our mom and dad's HLA genes to be different as this would allow us to present more possible antigens to our immune system.

There are also 3 genes that fall under HLA class II which are titled HLA-DP, HLA-DQ and HLA-DR. They also encode another 3 MHC proteins. These are the Class II proteins. These MHC II proteins are only used by immune cells. While the MHC I proteins are on every cell as a barcode of friend or foe, the MHC II proteins are only used by immune cells to present pathogens to the T helper cells to activate the adaptive immune response. With the MHC II genes you get a copy of each of these HLA-DP, HLA-DQ and HLA-DR from each parent. gives your immune cells 2 of each of these proteins with one coming from each parent. That means these immune cells will have a total of 6 MHC II proteins. Since they are cells, most of the immune cells present all 6 MHC I and all MHC II proteins.

The variation for each of these genes is vast. These genes have the most variation in the entire human genome. The HLA-A gene has over 4,000 known alleles. The HLA-B gene has over 5,000 known alleles. The HLA-C gene has nearly 4,000 known gene alleles. The HLA class II genes with HLA-DP, HLA-DQ, and HLA-DR have fewer possibilities, but they actually each have 2 genes that make each protein of the MHC II. One gene that encodes an alpha chain protein and one that encodes a beta chain protein. Those 2 chains come together to form the actual HLA protein. There are fewer alleles for these genes, but due to them having 2 actual genes for each of these MHC proteins, they have a lot of variation. There are over 12,000 different Haplotypes known for HLA. That means 1 in 12,000 people will have a match for their HLA haplotypes.

If you didn't think that was hard enough, the naming of each of these alleles for these genes doesn't get any easier. You will see a name like HLA-A*02:101:01:02N. Ya, that looks complicated, but we only need to understand the first few parts of these names to understand how HLA alleles work. The first part, the HLA-A, you already know as we just covered them. The part after the asterisk is the first key number. That is called the Field and it is the number for the allele of protein that gets coded. The numbers beyond that subdivide that field, but they are not as important. The HLA-A*02 is a key allele of the HLA-A gene as it is one of the most common to participate in rejection with T cells. When it comes to the HLA class II genes, you will see something like HLA-DPA and HLA-DPB for the alpha and beta chains that make up the HLA-DP protein.

When we talk about HLA alleles from parents, we might have HLA-A*02 from mom and HLA-A*04 from dad. That is how the allele of these genes are classified. This would be similar in the HLA-B and HLA-C genes with them followed by an asterisk and a number. Even the HLA class II genes are similar. You could have HLA-DPA*01 and HLA-DPB*02 from mom for one of your set of alleles and HLA-DPA*04 and HLA-DPB*01 for the other set. Just remember the MHC I proteins are only 1 protein made from 1 gene while the MHC II proteins are 2 chains from 2 genes that make up their proteins.

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Major Histocompatibility Complex I

We talked about the genes that encode the proteins that make up the Major Histocompatibility Complex (MHC). Now we will look at the proteins themselves. First we will look at the structure of the MHC I as it is the most important in rejection and makes up that barcode that identifies cells as friendly. There are 3 HLA genes that encode the 3 MHC proteins. They are HLA-A, HLA-B and HLA-C. The cell will make all 3 of these proteins. It will make them from both mom's chromosome as well as dad's chromosome. They might be different genes like HLA-A*02 and HLA-A*04, but the protein structures will have the same basic shape and functions.

The MHC I proteins are made up of domains linked together. Each domain is part of the alpha chain which is made up of 3 domains called the a1, a2 and a3 domains. There is a beta chain called the Beta 2 Microglobulin or B2M. The B2M chain is made by a common gene which makes the B2M protein. That protein gets used with every alpha chain. It is a generic protein that does not change. That means you will have genes to code the alpha chains, but they will all pair with a common B2M protein. This is an important concept and it distinguishes MHC I from MHC II which has genes for both the alpha and beta chains.

The MHC I has a structure that looks like a 4 leaf clover. The alpha 1 and alpha 2 domains would make up the top 2 leaves. The binding pocket sits right between the alpha 1 and alpha 2 connection. This is where the antigens bind. The bottom 2 leaves of the 4 leaf clover would make up the alpha 3 domain and the B2M that anchor the MHC I protein to the cell surface. The cell is constantly breaking down old proteins into fragments. The pieces from these proteins are called peptides. They bind into the pocket of the MHC I. Then they are carried to the cell surface and displayed for the immune system to see. These will be friendly protein fragments so the T cells will not respond. When the cell gets infected, some of the pathogen proteins will get broken down and displayed using the MHC I. Then the T cells will see those antigens and respond by killing the infected cell.

The antigens bind to the binding groove between the alpha 1 and alpha 2 domain. Each and every different allele of HLA-A would encode a different alpha chain which will recognize a different antigen in its binding groove. For example the HLA-A*02 you got from mom might recognize antigens with Leucines in it while the HLA-A*04 you got from dad might recognize antigens with Valine. This leads to diversity in our body for presenting antigens. The more variation we have in our MHC I proteins the more antigens we can possibly present to the T cells to mount a response. This also accounts for why one person might not respond well to a virus while another person might respond really well.

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Major Histocompatibility Complex II

Now we will look at the MHC II and how it plays an exclusive role within the immune system. This gets used by the Antigen Presenting cells (APCs). They use this MHC II to display peptides from proteins they break down and present them to helper T cells. The APCs make up the Macrophages, Dendritic Cells and B cells. They will engulf pathogens into their cytosol within these tiny bubbles called Phagosomes. They will take these phagosomes and merge them with lysosomes inside the cytosol that are filled with enzymes and Oxygen radicals. This will blast the pathogen with enzymes and chemicals that will break it down into tiny fragments. They will present these pathogens on their surface using the MHC II to display them for the helper T cells.

The MHC II comes from 3 sets of genes with HLA-DP, HLA-DQ and HLA-DR. There are two genes for each of the MHC II proteins with one making up the alpha chain and the other making up the beta chain. If we go back to our 4 leaf clover analogy, the alpha chain would have 2 domains and make up the left 2 leaves of the 4 leaf clover. The beta chain would also have 2 domains and make up the right 2 leaves of the 4 leaf clover. The binding groove for the antigen would sit right between the alpha 1 and beta 1 connection. The alpha 2 and beta 2 domains would be the parts that attach to the cell membrane. This is different from the structure of the MHC I which uses a single alpha chain and a common B2M beta chain. Here each chain is genetically different and plays an active role in making up the peptide binding groove.

There are 2 genes that encode each for the HLA-DP, HLA-DQ and HLA-DR proteins. They have an alpha and a beta chain. You will see HLA-DPA for the alpha chain and HLA-DPB for the beta chain. When it comes to naming the alleles of these genes, it will still include the asterisk and a number. You could see HLA-DQA*05 and HLA-DQB*02 for a set of alleles representing HLA-DQ. These alleles will encode a different peptide binding groove just like we saw with MHC I. The binding groove variety allows us to present more possible antigens to our immune system.

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Epitopes

This is a concept I want to include with MHC, but it applies to T cells, B cells, Antibodies and MHC. It is a bit of a complicated concept, but it is so critical for a biotech investor to understand. This is the concept of an epitope. Proteins are made up of amino acids. The amino acids get strung together into long strings. Those strings are often called peptides. Then they are folded into 3 dimensional shapes to form proteins or enzymes. The initial peptide can be any length of amino acids.

When it comes to the receptor of the immune cells, they can only recognize small fragments of these long strings of amino acids that make up these proteins. For T cells and MHC I, it tends to be about 7 to 10 amino acids long. For B cells, Antibodies and MHC II, it tends to be about 13 to 23 amino acids long. These small fragments of the larger protein that a receptor can recognize are called the Epitope. This is an important concept as a protein could be 500 amino acids long. You could have receptors that match many different parts of that larger protein. Each of them would be a different epitope. You could see dozens of possible T cells, B cells or MHC reacting to different fragments of this protein. This is important because every antibody therapy or cell therapy will target a specific epitope of a larger protein. They can each have dramatically different results. Some epitopes of a protein might even match other proteins which could cause off target effects.

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MHC Walkthrough

Here we are going to go deeper into the concept of the MHC and how it works. If you ever wondered why one person responded really well to an infection while the next person did not, it was probably related to MHC. If you ever wondered why some people have autoimmune disorders and other people do not, it is probably related to MHC. If you ever wondered why some people have successful transplants and others do not, it is probably related to MHC.

It all starts with the genes at the DNA level. You get 1 chromosome 6 from each parent for 2 copies of the number 6 chromosome. Each of these chromosomes will have a complete copy of the HLA genes. You will have and express both copies of each and every one of these genes as they are co-dominant. This means you expres 2 HLA-A, 2 HLA-B, 2 HLA-C, on every cell in your body. You will also have 2 HLA-DP, 2 HLA-DQ and 2 HLA-DR for each of your antigen presenting cells. Since the Antigen Presenting Cell is a cell, it will actually have both MHC I and MHC II proteins. Depending on your genetics, each and every one of the MHC proteins you make will be from a different gene allele. This will give you a broad variety of antigens you can present to those T cells to mount an immune response.

The MHC genes are actively expressed with cells constantly making new MHC complexes from every one of these genes. Every cell will make all of the MHC I proteins and display antigens on their cell surface for the Cytotoxic T cells. The Antigen presenting cells will make both MHC I and MHC II and present all of these antigens on their cell surface. Most of the time these will be self antigens which will not provoke a response.

When a cell becomes infected, it will begin to break down some of the pathogen's protein along with its own. These will get picked up with the MHC I and displayed on the cell surface. The Cytotoxic T cells work with the MHC I so they will see the foreign antigens in the presence of the MHC I. They will bind to these cells and release their granzymes and perforins which will kill the infected cell. The Antigen Presenting Cells will engulf and break down the pathogens they find in the fluids and tissues. They will break them down in their phagosomes and present the antigens on their cell surface. They will become active through their Pattern Recognition Receptors. They will leave the tissue and go to the nearest lymph node to present those antigens to helper T cells using MHC II. Because Antigen Presenting Cells can get infected, they can also process those MHC I antigens and display them to any Cytotoxic T cells in the lymph node when they present their MHC II antigens. This is called cross presentation.

The final concept of MHC is which cells use which. If you haven't noticed thus far, the MHC I and MHC II are used by different cells. They are also used with different T cells. The MHC I is used by each and every cell to present antigens from pathogens that infect them. They use this MHC I to present to the CD8 cytotoxic T cells. These T cells are designed to kill infected cells. They read that MHC I along with the antigen. They do not work with MHC II. The MHC II is used by antigen presenting cells to process pathogens they engulf and break down. They present these antigens to the Helper T cells in the lymph nodes using MHC II. The helper T cell does not recognize the MHC I. It only sees antigens in the presence of MHC II. This class I and Class II use of HLA genes and MHC separates the jobs of the T cells to specific roles. The Helper T cells work with the Antigen Presenting Cells and MHC II. The cytotoxic T cells work with all the tissue cells that might get infected using MHC I.

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HLA in Cell Therapies

The first thing we must understand about T cells is there are 2 kinds. The Helper T cells regulate the immune response and work with Cytotoxic T cells and B cells which are the effectors of the response. When we do cell therapies, the bulk of that therapy will be the cytotoxic T cells. This is because we need a lot of them to do the cell killing of the tumor cells. We only need a small amount of the helper T cells to help coordinate the response.

Normally, our resting immune system is almost all Helper T cells as they are the generals that control the immune response. We have low levels of Cytotoxic T cells as they do all the killing. When needed, the cytotoxic T cells expand to respond. The design of the Cytotoxic T cell is to find and kill infected cells or cancer cells. The Cytotoxic T cell uses a T cell receptor which works with only one HLA allele. It might might work with HLA-A*02 or HLA-A*04. You can see how this quickly becomes a problem when taking T cells from one person to make a therapy for another person. Those MHC I alleles might not match. Most of the time, they probably won't.

If you take a T cell that recognizes only HLA-A*02 and put it into a person with HLA-A*04, you can get rejection. That T cell will display all the MHC I antigens from the person who created it. The patient who receives it might have T cells that have different MHC I antigens. That patient's T cells would kill the donor T cell before it could even do its intended work and kill the cancer. This is why they immune suppress patients for cell therapy treatments. The other issue is that T cells won't recognize that patient's MHC I. I won't be able to work with the antigen being presented by the cancer cells of the patient. It can only recognize the exact HLA it was created for. Sometimes it can even get miss directed and respond to something it should not and cause off-target harm to the patient. That is extremely rare as T cells need 3 steps to activate them. Most of the result from a mismatch in HLA is ineffective cell therapies.

Due to this issue, most cell therapies approved today work from using the patient's own T cells to create the cell therapy. These are called autologous cell therapies. They take the T cells from the patient, and edit them to redirect them toward the cancer and put them back in. This works well, but has 2 challenges. The fist is many cancer patients have their immune system really beat up from chemotherapies. They don't have many good quality T cells left to make these therapies. The second challenge is that of time. It can take weeks to make therapies this way. That is time desperate patients don't have.

This has given way to research into overcoming the challenges of using allogeneic cell therapies. Those are the therapies that use cells from a healthy donor. Some of the engineering in these cells looks at removing the T cells receptor so it can't react with the patient's cells or MHC. They do this by knocking out the T cell receptor and replacing it with a modified B cell receptor that doesn't need MHC to work. The other is to remove the MHC I proteins from the donor T cell so that the patient's immune system doesn't reject those cells. We will get into this much deeper in cell therapies.

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