Immunoglobulins: Genes

Another post about immunoglobulins! This time it's about gene rearrangements, which I covered briefly for BIOC2001, but now it's time to go into more detail!

Describe multigene organisation of immunoglobulin genes

As you should know by now, antibodies are made up of two identical light chains and two identical heavy chains. Furthermore, these light chains can be either κ or λ. Heavy chains, κ light chains and λ light chains occupy different loci on different chromosomes. Each of these loci contain several gene segments that can combine in different ways to provide a wide variety of antibodies, as mentioned in one of my previous posts. These gene segments are the leader (L), variable (V), joining (J) and constant (C) sequences. Heavy chain genes also have diversity (D) sequences.

Before I get into how the genes are rearranged, I'll quickly run through how they are arranged to begin with.

λ light-chain genes have around 30 leader-variable sequence pairs (each leader sequence has a specific variable sequence that it's always paired with), as well as 4-5 joining-constant sequence pairs (every joining sequence has a constant sequence that it's always paired with).

κ light-chain genes are slightly different in that they also have leader-variable sequence pairs, but not joining-constant sequence pairs. Instead, there are around 5 joining sequences, which all combine with the same constant sequence, as the κ light-chain only has one constant region.

Finally, heavy-chain genes have around 40 leader-variable sequences (you don't need to remember the numbers, by the way), around 23 diversity sequences, around 6 joining sequences and 9 different constant sequences. Each constant sequence corresponds to a different kind of antibody, as mentioned in my previous post about immunoglobulins. Each constant sequence encodes all 3 or 4 domains of the constant heavy chain in question.

Describe variable region gene rearrangements
Describe the mechanism of Ig DNA rearrangements

In most cells of the body, the gene segments encoding antibodies are located far apart from each other. However, in cells that are going to grow up to become B-cells, an irreversible process called somatic recombination moves some of these gene segments closer together.

First, let's look at heavy-chain genes, because I'm pretty sure they get rearranged first. The first step that happens here is that a diversity (D) sequence gets joined to a joining (J) sequence. Just think of it as DJ, as in "It's murder on the dance floor, you'd better not splice this wrong, DJ!" (I'm sorry, I'm terrible at parody lyrics.) The next step is the splicing of a leader-variable pair with the DJ- the VDJ rearrangement. Both of these rearrangements are done courtesy of the RAG-1 and RAG-2 (recombination-activating gene 1 and 2) recombinases. After this, excess mRNA (most notably the bit between the last joining sequence and the required constant sequence) is spliced, a poly-A tail is added and the mRNA leaves the nucleus to be translated. As translation occurs, the L-sequence pulls the growing polypeptide into the lumen of the endoplasmic reticulum before being cleaved off. After all this you finally have a ready-to-go heavy chain!

Light-chain rearrangement is a little bit simpler because they don't have diversity sequences. The only rearrangement here is VJ, which is also catalysed by RAG-1 and RAG-2. After this it goes through all of the processing and translation steps, just like the heavy chain, before it is ready.

When both the light and heavy chains are done, they can then combine. Since there are a lot of different light chains that can be made and a lot of different heavy chains that can be made, there are an enormous number of different antibodies that can be made- and that's not even including some of the other ways in which variability can be introduced!

Another important thing to know about is that there are Recombination Signal Sequences (RSS) flanking the gene segments in both light- and heavy-chain genes. RSS can be one- or two-turns, depending on whether they make up one or two turns of the DNA helix. The rule here is that a one-turn RSS can only join with a two-turn RSS, thereby preventing improper joining of the segments. RSS have both conserved and nonconserved parts: generally a conserved nonamer (9 base pairs) and conserved heptamer (7 base pairs) with around 12-24 nonconserved base pairs in between.

Joining of the segments is not always the same each time. There can be a tiny bit of flexibility in where exactly the RAG-1 and RAG-2 enzymes cleave the DNA, leading to something called "junctional flexibility" that adds to antibody diversity. These rearrangements can be productive or non-productive: non-productive rearrangements have a premature stop codon somewhere in their sequence, while productive rearrangements don't.

One last VERY IMPORTANT note: The DJ, VDJ and VJ rearrangements that I've spoken about above are things that are only done ONCE in the B-cell lifespan. Hence a B-cell will make the same antibody for life. The antibody might change slightly due to mutations and so forth, but not dramatically. Also the antibody can become one of a different class (so IgM can become IgG as I'll discuss later), but it will still recognise the same antigen.

Describe class switching

As I just mentioned, an antibody can switch classes. All B-cells start off producing IgM, so in order to produce the other types of antibodies, they have to do something known as class switching.

Switching between IgM and IgD is relatively straightforward and also reversible as it doesn't actually change anything in the DNA. Essentially the location of the poly-A tail determines whether the antibody generated will be IgM or IgD, and if it will be secreted or membrane-bound. There are four important polyadenylation sites to know about: 1, 2, 3 and 4.

1. Poly-A site 1 is located right after the μ constant regions (IgM). Polyadenylation here causes formation of secreted IgM.
2. After poly-A site 1, there are two "membrane exons" called M1 and M2, required for insertion into the membrane. Poly-A site 2 is located right after these membrane exons, resulting in formation of membrane-bound IgM.
3. Poly-A site 3 is right after δ constant regions and thus results in secreted IgD.
4. Poly-A site 4 follows both the δ constant regions and the membrane exons M1 and M2 (yup, IgD has its own membrane exons).

"New" B-cells favour polyadenylation sites 2 and 4, so naïve mature B-cells will express membrane-bound IgM and IgD. (Immature B-cells express membrane-bound IgM only.) Again, since the generation of membrane IgM and IgD is completely controlled by where RNA is polyadenylated and spliced (a process known as "differential RNA processing"), cells can continue to express both IgM and IgD.

To get IgG, IgE and IgA, further rearrangements of the DNA need to be done. Since this involves splicing out some of the DNA permanently, these rearrangements are irreversible. Basically what happens is that the LVDJ of the heavy-chain gene combines permanently to a constant region other than the ones encoding μ (IgM) and δ (IgD). This occurs when B-cells are stimulated by an antigen, and the process is helped along by an enzyme called activation-induced cytidine deaminase, or AID. (So I guess you can say that the process is AIDed along. Ha. Ha. Ha. I'm sorry.)

The rearrangements of the DNA is facilitated by special regions known as switch sites. Each constant region, aside from IgD, has its own switch site. (IgD doesn't need one because switching from IgM to IgD is completely mediated by differential RNA processing, as described earlier.) When it's time for the DNA to be rearranged, the DNA loops around so that the switch site before the new constant region meets the switch site of the old constant region. The loop is excised, resulting in a shorter piece of DNA that now produces a new class of antibody.

Multiple class switching events can happen in the lifetime of a B-cell, but these events must always proceed in the "forward" direction. To better explain, let me quickly give you the order of constant region genes: μ, δ, γ3, γ1, γ2b, γ2a, ε and α. Any of these genes can be switched for anything to the right of it (well, except for δ, because IgD is a bit different). However, you can't go backwards in the list. Why? Well, when you switch from IgM to IgE, everything in between gets excised out. Hence you can't switch back to IgG, because none of the gamma genes are in the DNA any more.

Describe the generation of antibody diversity

Last point to go over for this topic! I've already touched on a lot of the ways in which antibody diversity is generated, but it's nice to have a summary.

1. There are a lot of genes that encode antibodies, and they can be combined in different ways (i.e. you can have one of 30+ variable sequences, 4-6 joining sequences etc.)
2. Junctional diversity- there's a bit of variation in where exactly RAG-1/2 cleave, resulting in some different sequences.
3. P-nucleotide additions- now here's something that I haven't touched on yet! When RAG enzymes cleave the DNA, this forms a "hairpin" structure in the cleaved ends. When these hairpins are cleaved in order to join up D-J and V-D-J/V-J, complementary nucleotides are added. These latter additions are known as P-nucleotide additions.
4. N-nucleotide additions- another topic I haven't touched on! Aside from P-nucleotide additions, sometimes an enzyme called TdT (terminal deoxyribonucleotidyl transferase- a real mouthful that makes you thankful for the acronym) adds in a few extra random nucleotides to seal up the gap. This adds in a bit more diversity in the binding site.
5. Somatic hypermutations- Just like any other gene, genes encoding antibodies are prone to mutation. However, the mutation rate is 100 000x higher than that in other genes. Mutations are most likely to occur in hypervariable regions, especially in activated B-cells in germinal centres. Somatic hypermutations are responsible for affinity maturation, or the progressive increase in affinity of an antibody for an antigen during the course of an immune response. (And yes, before you ask, some mutations can be detrimental and make an antibody lose its affinity for an antigen. This makes the B-cell less likely to receive survival signals, so "survival of the fittest" means that the better-performing B-cells live on to protect us better.) One last thing you need to know about somatic hypermutations is that they are usually associated with class switching, as both processes require AID enzymes.
6. Finally, as mentioned earlier, there are a wide range of H chains that can pair with a wide range of L chains, giving us a lot of combinations.

Whew! Another post down! There's just one more topic in the upcoming midterm that I haven't blogged about. (The midterm's next Monday- scary to think that it's already halfway through semester!)

Probability

This semester, I am doing Introduction to Statistics as an elective. (Yes, I'm a nerd. As if you didn't already know that.)

These posts will be a bit different in that I'm not going to go through the lectures, but instead I'm going to work through some of the practise problems. I'm not entirely sure if this breaches copyright but if it does, I'll take these posts down.

Next week we have a topic test on Modules 1-4. I'm going to work backwards from Module 4 to Module 1, so accordingly this post is actually on content from Module 4.

1. A 6-sided die is rolled.

a) What is the sample space for this experiment?
The "sample space" is simply a list of all possible outcomes, so the sample space for this is S = {1, 2, 3, 4, 5, 6}.

b) What outcomes are associated with the event E of "an even die roll is observed?
2, 4 and 6.

2. States and Outcomes

a) You flip a coin 2 times. How many states are possible for the outcome of this experiment?
There are four states: HH, HT, TH or TT.

b) You flip a coin 4 times. How many states are possible for the outcome of this experiment?
There are 2^4 = 16 states, that is 2 possibilities for the first coin multiplied by 2 for the second, 2 for the third etc. If you don't believe me, you can write out all of the outcomes, but that's a waste of time that you could be spending watching Netflix.

c) You roll a 6-sided die 3 times. How many states are possible for the outcome of this experiment?
On a similar vein to the previous question, the answer here is 6^3 (i.e. 6 possibilities for the first roll multiplied by 6 for the second, etc.)

d) You question 5 survey participants, and ask them whether they prefer coffee, tea or soft drinks. How many states are possible for the outcome of this experiment?
3^5 (3 possibilities for the first participant multiplied by 3 for the second etc. Be careful to do 3^5 here and not 5^3.)

3. Number of occurrences- Combinations

a) For 2 coin flips, how many ways can you get 1 or more heads?
For two flips it's relatively easy to just write out all the options, which are HH, HT, TH and TT. As you can see there are three ways to get one or more heads.

b) For 4 coin flips, how many ways can you get 1 or more heads?
Here the easiest thing to do would be to find out the number of ways to get 0 heads and subtract that from the number of total combinations. The number of total combinations is 2^4 = 16, and there's only one way to get 0 heads (TTTT). Hence there are 15 ways to get 1 or more heads.

c) When rolling 3 six-sided dice, how many ways can you get a total of 4 or less over the three dice?
This one you can also kind of write out. 1+1+1 = 3, 1+1+2 = 4, 1+2+1 = 4, 2+1+1 = 4. Hence there are 4 ways to get a total of 4 or less.

d) You question 5 survey participants, and ask them whether they prefer coffee, tea or soft drinks. How many ways can there be 3 or more "coffee" responses?
For this question, you can use the "n choose r" formula, which you might remember if you did 3CD maths in year 11/12. This formula is (n choose r) = (n!)/(r!(n-r)!).

Firstly, let's choose exactly 3 coffee responses from the 5 participants. This gives (5 choose 3) = (5!)/(3!2!) = 20/2 = 10
Now choose 4 from 5: (5 choose 4) = (5!)/(4!1!) = 5/1 = 5
And finally choose 5 from 5, which is obviously just 1 (all coffee): 1
The sum is 10 + 5 + 1 =16.

An alternative way of doing this would be to find the number of 2 or fewer "coffee" responses and subtract from the total number of responses, but that's too much effort.

4. Events. For each experiment and event of interest, identify how many states of the outcome are covered/included in the given event, what fraction of all possible outcomes are covered by the event and if possible, estimate the probability of the event.

a) You flip a coin 2 times, and you are interested in the event "one or more heads comes up."
As mentioned before, there are 3 states for this one: HH, HT or TH. As there are four outcomes total, 3/4 outcomes are covered by this event. Since all of these outcomes have the same probability, the probability of this event is also 3/4.

b) You flip a coin 4 times, and are interested in the event "one or more heads comes up."
Again, as mentioned before, there are 15 states for this and 16 total combinations. Hence 15/16 outcomes are covered by this event. Again, all of the outcomes have the same probability so the probability of this event is 15/16.

c) You roll a 6-sided die 3 times, and are interested in the event "sum of the dice is 4 or less."
As just mentioned, there are 4 states to this: 1 + 1 + 1, 1 + 1 + 2, 1 + 2 + 1 and 2 + 1 + 1. There are 6^3 possible outcomes, so the fraction of outcomes here is 4/(6^3) = 4/216 = 1/54. Again, all of the outcomes have the same probability so the probability of this event is also 1/54.

d) You question 5 survey participants, and ask them whether they prefer coffee, tea or soft drinks, and are interested in the event of "3 or more of the participants prefer coffee."
From my answers to previous questions, there are 16 outcomes in which 3 or more participants prefer coffee, and 3^5 outcomes total. Hence 16/243 outcomes are covered by the event. It is not possible to estimate the probability here because the probability is not uniform. People are not equally likely to prefer coffee, tea or soft drinks.

5. The blood groups of 200 people are distributed as follows: 50 have type A blood, 65 have type B blood, 70 have type O blood, and 15 have type AB blood. If a person from this group is selected at random, what is the probability that this person has O blood type?

P(O blood type) = 70/200 = 35/100 = 0.35.

6. The number of adults living in homes on a randomly selected city block is described by the probability distribution shown in the following table. What is the probability that 4 or more adults reside at a randomly selected home?

Number of adults, x	1	2	3	4 or more
Probability, P(x)	0.25	0.50	0.15	???

Since the total probabilities always sum to 1, P(4 or more) = 1 - (0.25 + 0.50 + 0.15) = 0.1.

7. If the probability of an event is p = 0.1, why might we see the event 14% of the time, or maybe only 9% of the time, after a small number of draws?

There are two important concepts to understand here: the Law of Large Numbers and sampling error. The Law of Large Numbers states that in the short-term you cannot predict the outcome, but after repeated trials the probability of an event converges around its "true" probability. The other important concept to know about is sampling error. This basically states that if you randomly select one sample and test it, you may end up with a different result if you pick another random sample and test that. This is because the samples are small, so the probability of certain events in them are not going to necessarily converge around the "true" probability value.

8. In the region around the Chernobyl nuclear disaster, mutated plants occur at a higher rate than elsewhere. Only 2% of the "normal" wild-growing chamomile plants have orange flowers, with the other 98% being white, but in the radioactive zone around Chernobyl the rate is 15% orange and 85% white.
If the region downwind of Chernobyl is sampled for seeds, and 25% of seeds collected are known to be from the radioactive zone, what is the probability that the seed will have orange flowers?

The seed will have orange flowers if a) it is one of the 2% of orange plants from a normal area or b) it is one of the 15% of orange plants from the radioactive zone.
P(normal, orange) = (0.75)(0.02) = 0.015
P(radioactive, orange) = (0.25)(0.15) = 0.0375
P(orange) = 0.015 + 0.0375 = 0.0525

9. Evaluate the following n choose x values.

a) 4 choose 1
(4!)/(1!3!) = 4

b) 4 choose 3
(4!)/(3!1!) = 4

c) 5 choose 2
(5!)/(2!3!) = (20/2) = 10

d) 6 choose 2
(6!)/(2!4!) = (30/2) = 15

10. Calculate the given binomial probability, and state what each means in words.

a) b(1; 4, 0.3)
This is the probability of getting 1 success out of 4 trials, with a 0.3 probability of success.
The probability here is (4 choose 1)(0.3)^1(0.7)^3 = (4)(0.3)(0.49)(0.7) = (1.96)(0.21) = 0.4116

b) b(3; 4, 0.3)
This is the probability of getting 3 successes out of 4 trials, with a 0.3 probability of success.
The probability here is (4 choose 3)(0.3)^3(0.7)^1 = (4)(0.09)(0.3)(0.7) = (0.36)(0.21) = 0.0756

b) b(2; 5, 0.4)
This is the probability of getting 2 successes out of 5 trials, with a 0.4 probability of success.
The probability here is (5 choose 2)(0.4)^2(0.6)^3 = (10)(0.16)(0.216) = 0.3456

b) b(2; 6, 0.4)
This is the probability of getting 2 successes out of 6 trials, with a 0.4 probability of success.
The probability here is (6 choose 2)(0.4)^2(0.6)^4 = (15)(0.16)(0.6)^4 = 0.31104

11. Suppose that the probability that a baby is a boy is 0.5 (and likewise for a girl). Which gender distribution is more likely: Family A, with 7 girls out of 8 children, or Family B with four girls out of 8 children?

Without even evaluating it I can see that Family B is more likely. There are only 8 different ways in which there can be 7 girls: the boy can be child 1, 2, 3, 4, 5, 6, 7 or 8. There are many more ways in which there can be 4 girls. Since the probability of a boy or girl is even, Family B is more likely.

12. Suppose that the probability that a lottery scratch ticket is a winner is 0.15.

a) What is the probability that a person buying 5 tickets will have no winning tickets?
P(no wins) = (1 - 0.15)^5 = 0.4437

b) What is the probability that a person buying 5 tickets will have at least one winning ticket?
P(at least 1 win) = 1 - P(no wins)
P(at least 1 win) = 1 - 0.4437 = 0.5563

c) Is it more likely that a person buying 5 winners will have at least one winning ticket, or have no winning tickets?

Well, if they buy 5 winners, of course they have winning tickets!

Assuming they just meant "buying 5 tickets" though, as you can see from a) and b), it's more likely that they'll have at least one win than no wins.

d) What is the probability that a person buying 10 tickets will have at least one winning ticket?
P(at least 1 win) = 1 - P(no wins)
P(at least 1 win) = 1 - (1 - 0.15)^10 = 0.8031

13. Use the Binomial Distribution formula to compute the following probabilities:

a) The probability of 1 or more heads in 2 coin tosses.
P(1 or more heads) = 1 - P(no heads)
P(1 or more heads) = 1 - (2 choose 0)(0.5)^0(0.5)^2 = 0.75

b) The probability of 1 or more heads in 4 coin tosses.
P(1 or more heads) = 1 - P(no heads)
P(1 or more heads) = 1 - (4 choose 0)(0.5)^0(0.5)^4 = 0.9375

c) The probability of 3 or more coffee responses in a survey of 5 people's favourite drink, if 60% of people usually prefer coffee.
P(3 responses) = (5 choose 3)(0.6^3)(0.4^2) = 0.3456
P(4 responses) = (5 choose 4)(0.6^4)(0.4^1) = 0.2592
P(5 responses) = (5 choose 5)(0.6^5)(0.4^0) = 0.07776
P(3 or more) = 0.3456 + 0.2592 + 0.07776 = 0.68256

14. The probability that a student is accepted to a prestigious college is 0.3. If 5 friends apply, what is the probability that at most 2 are accepted?

P(0 acceptances) = (5 choose 0)(0.3^0)(0.7^5) = 0.16807
P(1 acceptance) = (5 choose 1)(0.3^1)(0.7^4) = 0.36015
P(2 acceptances) = (5 choose 2)(0.3^2)(0.7^3) = 0.3087
P(2 or fewer) = 0.16807 + 0.36015 + 0.3087 = 0.83692

There's a whole lot more questions on normal distributions, but since a lot of them are of the "shade the part of the graph" kind, I'm not going to post about them here. Plus I'm getting sore just from sitting on this chair. TTFN!

Immunoglobulins: Structure and Function

Antibodies!

Be able to describe the basic structure of immunoglobulins (Ig)

I wrote quite a bit about this in one of my posts for BIOC2001, which also explains how hybridomas are made. (Hybridomas are immortal cells bred to produce monoclonal- i.e. all of the same kind- antibodies.) However, there's a bit more detail that you'll need to know for this course. I'm not going to go over the really basic stuff in the BIOC2001 post because I can't be bothered typing that out again, so if you know absolutely nothing about antibodies, please start there first!

Aside from the stuff I wrote on the BIOC2001 post, you also need to know that the light chains have a V_L (variable) and C_L (constant) domain, and the heavy chain has one variable V_H domain plus 3 or 4 constant domains (named C_H1-C_H4). Oligosaccharides are often seen N-linked to the C_H2 domain, allowing for greater stability and improved interactions with Fc receptors. IgG, IgA and IgD also have a "hinge region" at the area where the arms converge, which allows for some flexibility.

In the BIOC2001 post I also mentioned the Ig-folds (immunoglobulin folds) that make up the structure of each of the domains of the immunoglobulin. Well, it turns out that not only immunoglobulins have these. The immunoglobulin superfamily is a large group of cell membrane proteins that have at least one Ig-fold domain, and include T-cell receptors, MHC molecules, CD4, CD8 and CD3.

Another thing I mentioned in the aforementioned post (thanks Pattwood for sparing me having to type all of this again!) was the "papain cleavage site," where papain can eat an antibody, resulting in two Fab fragments and an Fc fragment. Well, pepsin can also be used to cleave antibodies, but in a slightly different place, or rather places as pepsin cleaves in more than one place. Pepsin cleaves a bit lower down, so the Fab fragments remain stuck together and are known as F(ab')₂. It also chops up the Fc fragment into multiple smaller ones. This chopped up Fc fragment is known as pFc'.

Be able to describe the biological activities of Ig

The most important thing to know here is that there are parts of antibodies that bind to antigen, as well as an Fc (constant) region that interacts with Fc receptors, resulting in other biological effects. Here are the most important effector functions of antibodies:

1. Neutralisation: Antibodies bind to toxins or microbes, which prevents said toxins or microbes from binding to receptors on the cell surface. This, in turn, prevents toxins from having their toxic effects.
2. Opsonisation: Binding to a microbe might make it easier for a macrophage or other phagocytic cell to eat it up.
3. Activation of complement: Antibody-antigen complexes can activate the classical complement pathway, which I'll discuss in a later post. This also leads to clearing out the invader.
4. Antibody-dependent cell mediated cytotoxicity (ADCC): NK cells can bind to antibodies which have become bound to a target cell. Cross-linking of Fc receptors signals the NK cell to release cytotoxic granules, killing the infected cell.

Be able to describe classes of Ig

There are five main classes of Ig: IgG, IgA, IgM, IgD and IgE (in order of abundance). Some of these also have subclasses: IgG has IgG1, 2, 3 and 4 and IgA has IgA1 and 2. They are named after the constant region of the heavy chain: γ, α, μ, δ or ε. All five classes can possess either a κ or λ light chain, with the κ chain being more common in both mice and humans.

IgG

IgG is the most common antibody, comprising around 80% of antibodies in serum. Its major activity is in plasma and extracellular fluids. IgG binds to FcγR (Fc gamma receptors), which allows for activation of complement and the ADCC pathway. IgG is unique in that it is the only antibody that crosses the placenta, allowing the foetus to benefit from passive immunity.

IgA

IgA is often found in secretions, such as tears, saliva, breast milk, digestive juices and so on. When in secretions, it is found as a dimer and associates with two other proteins: J-chain, which helps to hold the dimer together, and secretory component, which masks the protease cleavage sites. (When found in serum, however, IgA exists as a monomer.) IgA mainly helps by neutralising stuff so that the gut can push out the toxins via peristalsis. Unlike IgG, IgA doesn't activate complement and is unable to cross the placenta.

Secretory IgA gets into gut secretions by passing through epithelial cells located in intestinal crypts. These cells face MALT on one side and the gut lumen on the other. IgA in the MALT can bind to pIgR (poly Ig-receptor) on the MALT side of the epithelial cells. pIgR binds IgA as it passes through the cell in a vesicle. Once the IgA makes it to the other side, pIgR is cleaved and becomes the secretory component that associates with IgA and protects it from proteolytic cleavage.

IgM

IgM exists as a pentamer in serum, but as a monomer when docked in the B-cell membrane. Like IgA, it also has a J-chain, but it does not have a secretory component. IgM is the "first" antibody in the sense that it's the first to appear in phylogeny, the first to appear in the immune response and the first type that B-cells make during their development (I'll write more on this later!). They are largely confined to the blood and are good at agglutinating stuff (like blood from an incompatible blood type) and activating complement.

IgM is slightly different structurally from IgG and IgA. While IgG and IgA have only three C_H domains as well as a hinge region, IgM lacks a hinge region, but has an extra C_H domain to make up for it. C_H2 is essentially the surrogate hinge-region for IgM. (IgE is also a bit like this, as you shall soon see.)

IgD

IgD is kind of a mystery antibody in that we're not really sure what it does. It mainly exists on B-cell membranes, but it's not expressed until a B-cell becomes mature. Like most antibodies, it has three C_H domains and a hinge region.

IgE

IgE, as mentioned before, has four C_H domains but lacks a hinge region. It is normally found in only very low levels in the serum, which is good, because they're infamous for being involved in allergic responses due to their interactions with mast cells and basophils. Activation of these cells causes degranulation (i.e. release of histamine-containing granules), which results in many of the effects of an allergic reaction.

Be able to describe antigenic determinants on immunoglobulins

Antibodies have antigenic determinants, which according to my quick Google search, is really just another name for epitope. Yup, antibodies can bind to other antibodies.

There are three antigenic determinants:

• Isotypes: Differences in heavy and light chains that separate the classes and subclasses from each other. For example, γ, α, μ, δ and ε heavy chains and κ and λ light chains.
• Allotypes: Allotypes have the same isotype, but have small variations in the constant regions.
• Idiotypes: Idiotypes also have the same isotype, but have variations in the variable regions, causing them to recognise different antigens.

And that's the first three weeks of content down! Yay!

Cytokines and Mucosal Immunology

One of my friends majoring in immunology once gave the impression that immunology students see the word "cytokine" a lot. He wasn't wrong.

Cytokines

So what are cytokines? Well, they're pretty small molecules, but they're also pretty important molecules. They are messengers of the immune system that are secreted by many different cells of the body, most notably immune system cells such as macrophages, lymphocytes and so on. Types of cytokines include interleukins, chemokines, tumour necrosis factor (TNF), interferons (IFN) and so on.

Most cytokines act locally, via autocrine signalling (to the self) or paracrine signalling (through interstitial fluid to nearby cells). There are, however, some cytokines that act via endocrine signalling (via circulation). Cytokines have a very high affinity for their receptors, and so only nanomolar concentrations are needed to have an effect. Once bound, they can activate a variety of intracellular signalling cascades. These are important in a range of different situations, from immune responses, inflammation, haematopoesis, cellular proliferation, cell differentiation and wound healing.

Be able to describe cytokine signalling

Cytokines, as mentioned in my PHAR2210 post on enzyme-linked receptors, signal via the JAK/STAT pathway. Cytokine receptors are bound to Janus kinases (JAKs). When cytokines bind, the receptors dimerise, which brings pairs of JAKs close together. This allows JAKs to phosphorylate each other (autophosphorylation), as well as phosphorylate other areas of the cytokine receptors' cytoplasmic domains. All of this phosphorylation provides a nice environment for STATs (signal transducer and activator of transcription) to bind and become phosphorylated by JAK. Phosphorylated STATs are then able to form dimers and translocate into the nucleus, where they initiate gene transcription.

Be able to describe the roles of key cytokines

Unfortunately it looks like there's going to be a lot of details to memorise here, so bear with me.

Firstly, here's an overview of some of the cytokines that are going to come up through the course. This is by no means an extensive list- there's 35+ different kinds of interleukins (IL) for example, but you don't see all of them on this list.

• Proinflammatory cytokines: IL-1β, IL-6, TNF-α
• Anti-inflammatory cytokines: IL-10, TGF-β (TGF = transforming growth factor)
• T-cell related cytokines: IL-2, IL-4, IL-12
• B-cell related cytokines: IL-4, IL-5, IL-7
• Interferons: Type I and Type II

What they do is (mostly) pretty self-explanatory, but I'm going to go into a bit more detail and explanation anyway.

Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), as their name suggests, induce inflammation and activation of immune cells. They are made by most immune cells, particularly macrophages and dendritic cells. Unfortunately you can have too much of a good thing, and so if they're not properly controlled, they can release way too many cytokines- this is known as a "cytokine storm." Complications can arise due to the inflammation.

Anti-inflammatory cytokines (IL-10, TGF-β) are also pretty self-explanatory- they're mainly involved in shutting off and controlling the immune response. They can also activate immune cells that promote healing. The major producers of these are macrophages and T-cells. Just like pro-inflammatory cytokines, these can be problematic if too many are produced because then the immune response would be suppressed. The key here is balance between pro- and anti-inflammatory cytokines.

T-cell related cytokines (IL-2, IL-4, IL-12) function to support T-cell proliferation and activation. Some are made by T-cells, but not all: IL-2 and IL-4 are both made by T-cells (IL-4 by T-helper cells specifically- I think IL-2 is made by both helper and cytotoxic T-cells) but IL-12 is made by macrophages and dendritic cells.

B-cell related cytokines (IL-4, IL-5, IL-7) function to support B-cell proliferation and differentiation. So they're kinda like T-cell related cytokines, but for B-cells. IL-4 and IL-5 are actually made by helper T-cells, however, and play a large role in the allergic response. IL-7 is also not made by B-cells: instead it is made by stromal cells in the bone marrow, where it aids in B-cell development.

There are two main types of interferons: Type I IFN and Type II IFN. Type I IFN has two subtypes: IFNα and IFNβ. They are produced mainly by macrophages and dendritic cells, and help fight viruses. Type II IFN includes the subtype IFNγ, and is produced by T and NK cells in order to enhance immune responses.

Attributes of Cytokines

There are four main attributes of cytokines that you should know about:

1. Pleiotropy: The ability of one cytokine to be able to exert different effects on different cells.
2. Redundancy: Sometimes, multiple cytokines might all perform the same function.
3. Synergy: Cytokines can work together in order to cause something to happen (i.e. multiple different cytokines might be needed).
4. Antagonism: One cytokine might block the effects of another.

Cytokines in Mucosal Immune Responses

Cytokines can induce inflammation. They might do this, for example, if there's something wrong with your normal gut flora. If this isn't well controlled, however, this can result in a chronic inflammatory disease, such as IBD (Inflammatory Bowel Disease).

Sorry that that's such a shitty note to end on! (Yup, I just had to make that pun...)

Innate Immunity Receptors

Define how pattern recognition receptors function during an innate immune response

As mentioned in my first Immunology post, the innate immune system acts early, but non-specifically. That's not to say that there aren't any specific things that the innate system looks out for, however. There are a wide range of molecules that can be detected by receptors on cells involved in the innate immune response.

The main types of molecules that are detected by these receptors are called PAMPs and DAMPs. PAMPs are "pathogen-associated molecular patterns" and include some of the features that are common to microbes (but not to humans), such as lipopolysaccharide on the cell walls of gram-negative bacteria. DAMPs are "danger-associated molecular patterns" and are molecules that generally have some kind of usual function when they're within a healthy, functioning cell, but are released from infected or damaged cells.

So what are these receptors exactly? Broadly speaking, they are known as Pattern Recognition Receptors (PRR), and are expressed by many cells of the body (not just immune cells). These receptors are activated when bound, causing a cascade of other signals to happen. There are several different types of PRRs, as you shall see...

Describe features of different innate immune receptors

Toll-like receptors

Toll-like receptors (TLRs) are located on the surface as well as inside cells, and are able to recognise DAMPs and PAMPs. They are generally anchored to a membrane of some sort. On one side of the membrane they have a horse-shoe structure made of leucine-rich repeats (LRR) which assists in ligand binding. On the other side they have an intracellular cytoplasmic tail, which assists in signal transduction. They sometimes form homodimers or heterodimers.

Now it's time to look at a select few in a bit more detail!

TLR4 is located on the cell membrane and recognises lipopolysaccharide (LPS), which as I mentioned earlier is a component of Gram-negative bacteria cell walls. In order to do so, however, it requires some help from a couple of other proteins: CD14, which binds LPS and brings it to TLR4, and LPS-binding protein, which stabilises the interactions between LPS and TLR4. The cytoplasmic tail of TLR4 is associated with MyD88, which is an adaptor protein. Adaptor proteins are proteins that dock signalling proteins so that they're all nice and close and ready to signal. In the case of TLR4, signalling occurs via the NFκB pathway, which induces transcription of inflammatory cytokine genes. This ultimately results in cytokine expression, as well as expression of some cell surface molecules (such as CD80 and CD86) which are required for antigen presentation.

TLR7 is usually located in the nucleus or cytoplasm, but during infection with an ssRNA virus, it is located on the membranes of endosomes. Endosomes are the little bubbles that are made when things are endocytosed. In this case, it's the ssRNA virus that's being engulfed in the endosome. This exposes the ssRNA genome of the virus so that it can bind to TLR7. Ultimately, this leads to expression of Type I Interferon (IFN), which as I mentioned here, is not good from the virus' point of view.

Expression of IFN isn't the end of the game for a virus, however. While IFN can induce signalling pathways producing products that block virus replication and propagation, the virus can block the production of these products or even production of IFN itself. Rude.

NOD-like receptors

NOD-like receptors (NLRs) are so-called because they contain a nucleotide-binding oligomerisation domain, or NOD. That's a bit of a mouthful, but all you really need to know is that they're located in the cytoplasm and detect breakdown products of peptidoglycans, which are components of bacterial cell walls. Just like TLR4, NLRs can induce the activation of NFκB. (One thing to remember is that of the receptors in this post, those that target bacterial components induce NFκB activation, while those that target viral components induce IFN activation.)

RIG-I-like helicases

Wow, so many acronyms! RIG-I here stands for retinoic acid-inducible gene I. RIG-I-like helicases (RLH- yup, yet another acronym!) float around in the cytoplasm, where they bind viral RNA via their helicase domains. Just like TLR7 (which also detects viral RNA), activation of RIG-I results in induction of type I IFN production.

Antigens and Immunogenicity

Onto another post!

Describe the properties of an immunogen vs. an antigen

Let's start this off with some definitions!

• Immnogen: A substance that can induce an immune response. All immunogens are antigens.
• Antigen: A molecule that can react with a specific antibody and/or can be degraded into peptides recognisable by T-cells. Not always immunogenic.
• Epitope: The portion(s) of an antigen that can be bound by an antibody or a T-cell receptor. You may also think of these as the immunologically-reactive regions of an antigen.
• Valency: The number of things that an antigen or an antibody can bind at the same time. Antibody valency is the number of antigens that an antibody can bind (so your typical Y-shaped antibody will have a valency of 2), while antigen valency is the number of molecules that an antigen can bind to.

A quick note about stuff that's recognised by T-cells: while antibodies will recognise antigens in their native form (i.e. what they usually look like), T-cells are really fussy and will only recognise antigens once they've been processed and presented to them on MHC (major histocompatibility complex) molecules. Kinda like toddlers who will only eat vegetables if it's been disguised to look like junk food and is zooming into their mouth like an aeroplane.

Describe the factors contributing to immunogenicity

Immunogenicity is the ability of an immunogen to stimulate an immune response. Immunodominant epitopes are more immunogenic (i.e. induce a stronger immune response).

There are several different factors contributing to differences in immunogenicity:

• Foreignness: The more "foreign" a molecule is, the more immunogenic it is. Isogenic/autologous/self molecules are obviously the least immunogenic, followed by syngeneic (twins), allogeneic (different individual of the same species) and finally xenogeneic (someone of a different species).
• Molecular size: Generally, larger immunogens are better immunogens.
• Haptens: As mentioned in an earlier post, these are small molecules that are antigenic, but not immunogenic unless it's stuck to something else.
• Adjuvants: Adjuvants are substances that can enhance the immunogenicity of antigens despite not being immunogenic themselves. These are sometimes used in vaccines, such as Gardasil (the vaccine against HPV).
• Host biological system: In order to produce an immune response, the host must actually be capable of producing an immune response, i.e. not immunocompromised in any way.

Another point to make is that different cell populations might be involved depending on the route of exposure to an antigen. If exposure occurs subcutaneously or intramuscularly, the main response will be in the lymph nodes. If exposure is intravenous, there will be a general (i.e. systemic response), especially in the spleen. Finally, if exposure occurs intranasally or orally, the MALT of the mucosal membranes of these organs are likely to be involved.

Describe B-cell and T-cell receptors

B-cell receptors are essentially just membrane-bound antibodies. (Of course, antibodies can also exist in secreted form). B-cell receptors/antibodies, as I mentioned earlier, will recognise epitopes on antigens in their native form. An epitope can be made up of sequential amino acids, or discontinuous amino acids that are in the same area after protein folding.

It's important to note that antigen-antibody binding is non-covalent. That is, all of the bonds between antibody and antigen are hydrogen bonds, Van der Waals forces, and so on (see this early post for more info). For this to work, it is important that the antibody binding site has a complementary shape to the epitope that it's going to bind- the so-called "lock-and-key fit." Because of this, antibodies will show greater specificity for some antigens than others.

There isn't too much that we need to know about T-cell receptors at this stage, other than that they can only bind antigens that have been processed and presented to them.

Cells and Organs of the Immune System

Back to blogging about Immunology!

Describe the cells of the immune system and their function

I've already mentioned some of the immune system cells in an earlier post, but I'm going to give a refresher on those cells as well as introduce some others.

• Granulocytes: so-called because they have granules that can be seen under the microscope. All of these are found in the circulation. There are three main types:
• Neutrophils: First to arrive on the scene. Phagocytose microbes.
• Eosinophils: Phagocytose antibody-coated parasites. Also play roles in allergic responses.
• Basophils: Also play roles in parasite immune responses, allergies and inflammation.
• Mast cells: Granular cells found in the tissues. There is some debate over whether these are considered to be granulocytes or not. These also have roles in allergies and parasite expulsion.
• Monocytes and Macrophages: Monocytes exist in the circulation, but once they enter the tissue they can differentiate into macrophages. Both monocytes and macrophages play roles in phagocytosis. They also play roles in antigen presentation (well macrophages do at least, not so sure about monocytes).
• Dendritic cells: One of the main antigen-presenting cells (i.e. cells that process and present antigen to T-cells, which you'll meet later). In this way, they form a "bridge" between innate and adaptive immunity. They have different subtypes, classified mainly according to where they're found.
• Langerhans cells: found in epidermis and mucosal membranes
• Interstitial dendritic cells: found in most organs
• Interdigitating dendritic cells: found in T-cell areas of secondary lymphoid tissues and in the thymus
• Circulating dendritic cells: found in the blood and lymph
• Follicular dendritic cells: found in the B-cell rich follicles. These are a bit different because they do NOT function as an antigen-presenting cell. Instead, they express high levels of complement and Fc receptors, allowing them to bind immune complexes (a fancy name for optimised antigens- I've mentioned optimisation here) and store them, which in turn might help facilitate B-cell activation.
• Lymphocytes: These come in two main types- B cells and T cells.
• B-cells: Make antibodies (humoral immunity).
• Plasma cells: Specialised B-cells that secrete antibodies.
• Memory cells: Help the body "remember" an antigen.
• T-cells: Have different subtypes with different functions:
• Cytotoxic T-cells (CD8): Kill infected cells (cell-mediated immunity).
• Helper T-cells (CD4): Mediate the immune response.
• NK cells: Cells of the innate immune system that help to kill stuff.
• NK T-cells: A subset of NK cells that also have T-cell properties.
• Platelets: These aren't really whole cells, but rather small fragments of megakaryocytes. They can release inflammatory mediators and are involved in clogging up wounds and releasing stuff that aids in wound repair.
• Red blood cells: Not sure if these are really part of the immune system, but they're on the slides so I'll include them anyway. These transport oxygen around the body.

All of the cells in the blood are formed during a process called haematopoiesis. The main pluripotent stem cells involved here are called haematopoietic stem cells. They can further differentiate into two main types of progenitors: myeloid and lymphoid. These progenitors can then further differentiate into other types of cells. Lymphoid progenitors tend to differentiate into NK cells, T-cells and B-cells, whereas myeloid progenitors tend to develop into the other types of blood cells. Dendritic cells are a bit of a wildcard because they're thought to be able to develop from either progenitor, and also from monocytes.

Describe the organisation of the lymphoid system

The lymphoid system, just like so many other systems of the body, are made up of organs which are joined by connecting vessels called lymphatics. The lymphatics transport lymph, which is basically derived from the interstitial fluid of tissues. Lymphatics join up until they form the thoracic duct, which dumps its contents into the right brachiocephalic vein, effectively returning everything to the circulation. (More info on lymph vessels here.)

Explain the different roles of the primary and secondary lymphoid tissues

The primary lymphoid organs (bone marrow and thymus) are the main organs involved in the haematopoiesis of immune cells. All blood cells originate in the bone marrow, and the thymus (located in the anterior mediastinum) is where T-cells complete their development. The secondary lymphoid organs, which include lymph nodes, the spleen and mucosa-associated lymphoid tissue (MALT), are mainly involved in trapping antigens that enter the body through various routes. Let's look at some of these organs in more detail!

Lymph nodes

Lymph nodes are mainly involved in trapping local tissue antigen. Their structure looks somewhat like a kidney turned on its side. Incoming (afferent) lymphatics enter the lymph node on one side and outgoing (efferent) lymphatics leave on the other, after being filtered. Blood vessels also run through the lymph node, including high endothelial venules (HEV) which allow naïve lymphocytes to leak out and enter the lymph node.

The inside of the lymph node can be broken down into several compartments. The outer cortex contains follicles, which is pretty much the B-cell zone of the lymph node, but macrophages and follicular dendritic cells hang out there too. Germinal centres, which are regions of intense B-cell activation, also develop within the outer cortex.

Further inside the lymph node is the paracortex. This is the "T-cell zone" of the lymph node, but interdigitating dendritic cells hang out here too.

Even deeper inside is the medulla. This is where plasma cells hang out. (And yes, I recognise that plasma cells are B-cells, and these ones aren't in the B-cell zone. So maybe I lied about the outer cortex being a B-cell zone.)

Spleen

While the lymph nodes filter lymph, the spleen filters blood. Fun fact: in one day more lymphocytes pass through the spleen than all lymph nodes combined.

The spleen has lots of bits and pieces that you need to know about. Trabeculae provide the main structural support of the spleen. Blood is supplied to the spleen via the splenic artery, which splits into arterioles which end in vascular sinusoids. Blood then exits via the splenic vein.

The two main compartments of the spleen are the red pulp and white pulp. They're kinda easy to remember because the red pulp deals with red cells (it removes the old and defective ones) and the white pulp deals with white cells (specifically lymphocytes, which are activated here). The two compartments are separated by the marginal zone, which is where blood-borne microbes and antigen are trapped.

Dendritic cells transport antigen from the marginal zone to another area called the PALS. PALS stands for periarteriolar lymphoid sheath. The PALS surrounds the arteriole and mainly contains T-cells, which become activated when antigen is presented to them.

Just like lymph nodes, the spleen also contains follicles which contain mainly B-cells, as well as macrophages and follicular dendritic cells. The B-cells here are mainly activated in response to signals from T-cells.

Mucosa-Associated Lymphoid Tissue (MALT)

MALT protects the mucosa, as the name suggests. It can be further categorised into BALT (bronchial-associated lymphoid tissue), GALT (gut-associated lymphoid tissue) and so on. MALT actually makes up the bulk of lymphoid tissue.

MALT isn't just one big messy thing covering our epithelial surfaces. It actually does have some degree of organisation, as seen in the Peyer's patches in the ileum. MALT has special cells called M-cells, which deliver antigen to underlying immune cells. One last thing to know about MALT is that it is biased towards IgA production (as opposed to other kinds of antibodies) and has some specialised lymphocytes called mucosal homing receptors.

And that's it for that lecture! Lots of facts to remember...