a bonobo humanity?

Just something to start with…

It was in 1796 that Edward Jenner created the first vaccine, using material from a cowpox sore on a milkmaid’s hand and injecting it under the skin of an eight-year-old boy. I’ve not been able to ascertain whether Jenner injected the material directly into the boy’s bloodstream. In any case this has been regarded as the world’s first vaccination, a term coined by Jenner (from vacca, Latin for cow). Variolation, using smallpox scabs inhaled by the patient, had been practised in Asia for centuries before Jenner, and was promoted in Britain by Mary Wortley Monagu earlier in the 18th century. Variolation was certainly effective, and for some time there was a dispute about the best treatment. In any case, what was being brought into action by both treatments was the adaptive immune system.

So now to MIT’s introduction to immunology course, presented by Adam Martin. He mentions, in his first lecture, that there are many levels of immunity, and goes on to describe two, innate and adaptive immunity. Innate immunity is ‘the first line of defence’, and acts immediately. Neutrophils, which are white blood cells, are part of this innate system, which is quite static and unchanging, involving a constant surveillance. Adaptive immunity is also known as acquired immunity – and its acquisition takes time.

The adaptive immune system is more specific than the innate. That’s why we need regular flu vaccinations, because flu viruses can evolve quite rapidly. These vaccinations are designed to combat or provide immunity to new strains of the virus.

There are two branches of adaptive immunity: humoral immunity, which is protein-mediated (the proteins are called antibodies). This type of immunity is called humoral because these antibodies are secreted into bodily fluids or humors (blood, mostly). The types of cells that produce these proteins, these antibodies (Ab), are called B cells, which are matured in the bone marrow. The other branch is cell-mediated immunity, which involves T cells, which are matured in the thymus gland, near the top of the lungs.

So far so simple, but next we’re shown a scarily complex chart showing the derivation of all these cells from hematopoietic stem cells – most of these cells comprising the innate system, but also the T and B lymphocytes of the adaptive system (and they’re called lymphocytes because they’re the primary cells found in the lymph). So, it’s important that we can trace the ‘tree’ back to its ‘roots’, the progenitor cells.

Both the humoral and cell-mediated immune system cells have antigen receptors, which recognise specific antigens (Ag). An antigen is any substance that creates/generates an immune response.

Antibodies – goodies; antigens – baddies.

So, the B cell antigen receptor is also called an antibody, and an immunoglobulin (Ig). Structurally, antibodies are proteins with a lipid bilayer, which represents the plasma membrane. Outside of this membrane is the exoplasm, and inside is the cytoplasm. (I’m a little confused here – this sounds like the description of a cell, not a protein). Anyway we’re describing a B cell, and the antibody (protein) can have a trans-membrane domain spanning the plasma membrane, and an exterior Ig domain. These domains are modular folds (polypeptide chains) that are separate from the rest of the protein, and are inserted into the cell membrane, with an N-terminus exterior to the cell and a C terminus within the cell. Each antibody protein has two long polypeptides, the identical ‘heavy chains’ of the molecule. They also have smaller ‘light chains’, and it’s all laterally symmetric. The external tips of these antibodies are what recognises and binds to the antigen.

Now to the T cell receptor (TCR). It’s very different, and simpler, structurally. It has two chains, alpha and beta, and fewer immunoglobulin repeats. The tip of this double chain interacts with the antigen.

The B cell’s receptor, or antibody, has different forms. Some antibodies have transmembrane domains and are anchored in the plasma membrane, but other forms lack the transmembrane domain and instead of being an integral membrane protein, they’re secreted into the blood. The membrane-bound form comes first, but as an infection progresses, the secreted form becomes dominant. The T cell receptor only has the membrane-bound form.

There are many different antibodies, and each given antibody will recognise a different antigenic structure. They can recognise small molecules, proteins, DNA, carbohydrates, lipids, etc. But apparently that’s the B cell’s antibodies. The T cell receptor is more restricted, recognising peptides – short sequences of amino acids – which are presented by the MHC complex (classes 1 and 2). More of that later.

Properties of the immune system. First, specificity – they can finely discriminate between molecules. This is of course essential to avoid the prospect of auto-immune diseases. So, how are such levels of specificity achieved? Well, it’s complex.

Looking at B cell antibodies again, with their heavy and light chains. There are different domains on the heavy chain that are either variable or (more or less) constant. And the same goes for the light chain, with the same lateral symmetry described above. So, looking at the amino acid sequence of the variable part of the antibody (protein) molecule, looking at its structure from N to C terminus …. (?)

By convention, peptide sequences are written N-terminus to C-terminus, left to right (in LTR writing systems) – [from Wikipedia, N-terminus]

Imagine taking these antibodies from the variable region of the chain and aligning them (their amino acid sequence) from N to C. Each is a different antibody (or heavy chain polypeptide) produced by a unique B cell. Then we consider the residue number… 

The amino acid residue number refers to the spot in the linear chain where that particular amino acid is found. For example, the number 20 means that amino acid is 20th in the chain.

.. and how much each amino acid residue varies along the sequence. ‘So if we were to align antibody gene stretches like this’, and check out the variation, we could graph it as in the diagram:

The Y axis being the amount of variation, and the X axis is the residue number along the polypeptide sequence. So you see from this graphic that there are three regions of hyper-variability. These regions are also called complementarity-determining regions (CDRs). Apparently there are always three of them?

So what are these regions? We’re shown a crystal structure of one of them (both heavy chain and light chain), with an antigen attached, as well as a ribbon diagram with its CDR, which contacts the antigen. We’re next shown an Ig fold with three loops extending from it, which can bind to external particles. These IG loops vary in amino acid sequence and each tiny variation will affect the binding ability, or affinity to an antigen. So the key here is the CDR.

Each B cell expresses a unique antibody protein, with unique specificity, due to its unique sequence at the CDR region. To get more of a particular antibody, the cell that expresses it can be clonally expanded, creating monoclonal antibodies. Each of those B cells will have an antibody, or antigen receptor, with that same specificity.

So that’s the generation of specificity, but the generation of diversity is also vital for enhancing the immune system. How is this diversity achieved? There are millions of B cells that have unique antibodies, and there aren’t a million genes producing those cells – we have about 30,000 genes. The answer is somatic ‘reshuffling’ or recombination. We have a single heavy chain gene, and two light chain genes, for antibodies. They’re made up of multiple gene segments. Specifically, the parts that make up the variable domain are composed of gene segments that are shuffled during the development of the B cell to give rise to a diversity of proteins.

So we’re next shown a graphic headed, no doubt importantly, ‘the immunoglobulin gene contains multiple V, D and J segments – one of each needs to be brought together to form a functional antibody gene’. The graphic, which I won’t reproduce here, shows the human Ig heavy chain locus on the right. On the left is a variable (V) gene segment, containing 45 variable genes. There’s a diversity (D) segment next to it, containing 23 more of these genes, and then there are 6 joining (J) segments. All of these are distinct regions of the gene, the exon that encodes this variable region of the antibody.

So there are multiple V, D and J segments.To generate a functional antibody, one V has to be brought together with one D, which then has to be brought together with one J to create the heavy chain. So the next graphic has the heading ‘Rearrangement of genomic DNA is needed to put together a gene that encodes an immunglobulin’. It indicates that, for the light chain, only V and J gene segments are required, whereas the heavy chain also requires the D segments. Most of the cells in our body, and also in the germline at the earliest stage of development, have this arrangement. But during lymphocyte development a recombination event occurs which brings the two (V and J), or three (V, D and J) gene segments together. So, recombination at the heavy and light chain genes for the antibody.

This is all quite different from the recombination that occurs during meiosis and the formation of the gametes. That recombination occurs between homologous chromosomes, while this type brings together and deletes segments along one chromosome to bring these V and J segments together (intra-chromosomal recombination) to create an antibody protein. It’s called VDJ recombination, and is specific to lymphocytes, because during the development of B and T cells there is an induction of recombinases that mediate this recombination [?] In this case a recombination mediated by ‘recombination-activating genes 1 and 2’, aka RAG1 and RAG2.  All this is lymphocyte-specific, and these recombinases mediate this rearrangement, bringing unique V, D and J segments together. The diversity derives from the fact that each segment has a unique sequence, coding for a unique amino acid sequence, and a distinct protein.

But the body derives further diversity from another process. When these segments are shuffled there is imprecision – nucleotides can be added or subtracted (deleted) when these segments are joined. This generates more amino acid diversity, called junctional imprecision. So we have imprecise recombination, leading to the insertion or deletion of nucleotides [the basic structural units of nucleic acids – RNA & DNA – consisting of a nucleoside and a phosphate group]. If there is a multiple of three nucleotides either inserted or deleted, the result is a functional antibody. A multiple of three is required, to avoid a ‘frameshift mutation’, because ‘a cell reads a gene’s code in groups of three bases when making a protein’. That’s the system upon which it operates – if you inserted/deleted a single nucleotide along the line you wouldn’t get a functional protein.

Another important thing – which happens not as a consequence of this recombination process, but of activating the T cell, which is that in addition to these variations, there’s also somatic hypermutation  – an elevated mutation rate at the Ig locus, which increases further the diversity of the amino acid at these variable antibody regions. This is also known as affinity maturation, as it increases the affinity of the antibody for its antigen. Note that this is T cell-mediated.

So the immunoglobulin gene is not expressed until this recombination occurs. So this recombination leads to the expression of either the heavy chain or the light chain gene. This is because the enhancer is downstream in the gene, so by vanquishing the intervening sequence you bring the promoter within range of the enhancer, and the gene is expressed. It’s really quite romantic.

Remember though that there are two copies of these genes – paternal and maternal, so there’s another feature of this system – allelic exclusion. A B cell expresses only one antibody, so if both alleles are expressing, that wouldn’t be the case. With allelic exclusion, if you get a recombination event that leads to a functional antibody for one of your inherited copies of the gene, it suppresses recombination on the other allele, so you will only get one heavy chain and one light chain gene expressed per B cell.

So these junctions between V, D and J segments are found in the CDR-3 region, and are responsible for the high level of variability at the CDR or hyper-variable 3 region.

So the last feature to describe is ‘memory’, the ability to recall a previously experienced infectious substance. The immune system needs to be able to do this, and this is the principle behind vaccination. A vaccine will inject an attenuated or inactivated foreign agent into your bloodstream so that the immune system will be alerted to that antigen if/when it turns up in the system later.

Several ways in which this ‘remembering’ manifests itself, comparing a primary infection with a secondary infection, the adaptive immune system has very different responses. The primary response is a little delayed, taking between 5 and 10 days, while the secondary response is generally between 1 and 3 days. The magnitude of the response, the concentration of antibodies that are produced, is larger the second time around. And the antibodies themselves have become ‘better’. This can be shown by antibody affinity – how tightly the antibody recognises (and binds to?) the antigen. Antibody affinity (‘tightness’ of recognition) is measured as the dissociation constant for an antibody to a given antigen. The lower the number, the tighter the binding. For the primary infection the antibody affinity is weaker, on the order of 10 to the seventh molar in terms of KD [KD, or K_D, is a quantitative measure of antibody affinity – ‘the equilibrium dissociation constant between the antibody and its antigen’], and the secondary infection generates antibodies that are functionally better, less than 10 to the negative eleventh molar (sub-nanomolar), a very tight interaction between two molecules. So, more and better. So from the first infection to the next, a type of B cell – a memory B cell – will express an antibody specific to the previously experienced antigen, because, due to irreversible recombination, this will be encoded in the genome. The memory results from VDJ recombination being irreversible, so those memory B cells will be there even if the antigen is not. These cells can be generated by vaccination.

So ‘effector functions of antibodies’. They can bind to a foreign substance, interfering with its functionality. Neutralisation, for example, which means preventing the antigen from entering cells. Also phagocytosis, recruiting phagocytes to internalise the antigen (e.g. a bacterium), and recruiting killing cells to kill cells.(natural killer cells).

The lecture ends with a story in the fight against breast cancer, a treatment based on a mouse monoclonal antibody. So, we can use antibodies from other species to generate treatments. One example is herceptin, which can be used as a treatment for HER2-positive breast cancer. It’s based on a mouse antibody which recognises the HER2 growth factor receptor, which is over-expressed in about 30% of human breast cancers. Researchers have engineered a human antibody with the mouse sequence at its complementarity-determining region. So you have a human antibody, which won’t be attacked or removed by the human immune system, but will recognise HER2, and recruit immune cells to HER2 positive cells, neutralising and possibly killing them. This is the possible therapeutic aspect of antibodies, whatever their source.

So ends the first lecture.

References

30. Immunology 1 – Diversity, Specificity, & B cells, MIT OpenCourseWare video

https://en.wikipedia.org/wiki/N-terminus#:~:text=the%20C%2Dterminus.-,When%20a%20protein%20is%20translated%20from%20messenger%20RNA%2C%20it%20is,end%20of%20the%20growing%20chain.

https://study.com/academy/lesson/what-is-amino-acid-residue.html#:~:text=The%20amino%20acid%20residue%20number%20refers%20to%20the%20spot%20in,is%2020th%20in%20the%20chain.

https://www.genome.gov/genetics-glossary/Frameshift-Mutation#:~:text=A%20frameshift%20mutation%20in%20a,bases%20when%20making%20a%20protein.

https://www.abcam.co.jp/primary-antibodies/kd-value-a-quantitive-measurement-of-antibody-affinity