IMI 3: The Adaptive Immune System

Question 1

Observe the learning outcomes of this session

Answer 1

Question 2

What are the two key features of the adaptive immune response?

Answer 2

• the immune system changes (adapts) to recognise pathogens specifically
• they memorise their characteristics, allowing the immune system to react much more quickly and specifically in the future

Question 3

The adaptive immune system has both humoral and cell-mediated components

Define humoral and cell-mediated components of adaptive immunity

Answer 3

• humoral components of adaptive immunity:
• antibodies that circulate in the blood, perfuse tissues and are secreted onto mucosal surfaces
• cellular components:
• B and T lymphocytes: have the ability to recognise antigens through receptors expressed on their cell surface

Question 4

Where do conventional B and T cells originate from?

Where do these mature lymphocytes then go?

Answer 4

• they originate in the primary lymphoid organs (lymphopoiesis)
• T cells: thymus
• B cells: bone marrow
• mature lymphocytes migrate to the secondary lymphoid organs and traffic around the body through blood or lymphatic circulation
• secondary organs include spleen and lymph nodes

Question 5

How much of leukocytes do B and T cells account for in blood?

How much of the cells do they account for in the lymph

Answer 5

• They account for 20-40% of leukocytes in blood and 99% of cells in lymph.

Question 6

How do B and T cells become mature?

What do they become?

Answer 6

• until encounter with an antigen, both B cells and T cells are naive
• upon the encounter with an antigen, they get activated and can become effector and/or memory cells

Question 7

Give an overview of B cells

• what they make
• naive form and mature form
• roles

Answer 7

• B cells make immunoglobulin molecules that bind highly specifically to foreign antigens
• each B cell produces a unique immunoglobulin, with a narrow specificity
• a diversity of immunoglobulin across all B cells of the immune system allows B cells to specifically bind to a wide range of antigens
• Naive B cells express a membrane-bound form of immunoglobulin called the B cell receptor (BCR)
• upon activation, they also produce the immunoglobulin in its secreted form, antibodies
• effector B cells that produce only antibodies but lack a BCR are called plasma cells
• B cells can also act as antigen-presenting cells
• they express phagocytic receptors that similarly to macrophages and dendritic cells, can internalise, process and present antigens on MHC II molecules to T cells
• they can also capture and internalise antigens through the BCR
• this is important because upon presentation, B cells receive signals back from the T cells and from the BCR that allow their differentiation into plasma or memory B cells

Question 8

Give an overview of conventional T cells

• what characterises them
• sub-classifications

Answer 8

• Conventional T cells are characterised by the presence of T cell receptor (TCR) and certain co-receptors (CD3 alongside either CD4 or CD8) that define their effector functions.
• The TCR can “recognise” (ie specifically bind) antigens but only when the antigen has been chopped up and is held by a MHC (major histocompatibility complex) molecule. T cells can be sub-classified as:
  1. T helper cells (with CD4 on their surface) have important functions in supporting other cells of the adaptive immune system.
  2. Cytotoxic T lymphocytes (CTL, with CD8 on their surface) that specialise in killing defective (infected; cancer) host cells. They release cytotoxic granules containing granzyme and perforin that can very effectively kill target cells.

Question 9

What is the adaptive molecule of B cells?

What are its two forms?

Answer 9

• the adaptive molecule of B cells is immunoglobulin (Ig)
• it comes in two forms:
  1. A transmembrane form that studs, or dots, the surface of the B cell.
• This is called the B cell receptor (BCR).
  2. A version of the BCR that is missing the transmembrane domain, but has a signal peptide instead, so it is secreted from the cell.

Question 10

What is a soluble Ig?

Answer 10

• it is called an antibody

Question 11

What is the immunoglobulin domain?

Answer 11

• In discussing the structure of immunoglobulin, we will mention immunoglobulin (the whole BCR or antibody molecule) and Ig domains.
• An Ig domain is a smaller chunk of the immunoglobulin molecule which has a conserved structure held together with an internal disulphide bond.
• The secondary structure of these domains is called the immunoglobulin fold.
• Ig is made up of four polypeptides each made up of 2-5 Ig domains
• This core structural motif is also found in many other proteins: proteins containing this fold (including Ig) are members of the immunoglobulin superfamily.
• In addition to Ig, the immunoglobulin superfamily includes many cell surface molecules you will encounter throughout IMI.
• For instance, this includes MHC molecules; CD4 and CD8; ICAM1; IL-1 receptor (IL-1R); some Fc receptors; inhibitory and stimulatory molecules such as CTLA-4 and CD28.
• Take care not to get confused between immunoglobulin (particularly the B cell receptor) and the immunoglobulin superfamily receptors.

Question 12

What is human Ig formed from?

How many genes do we have to code for human Ig?

Answer 12

• human Ig is formed of four polypeptide chains:
• two identical heavy chains
• two identical light chains
• these are connected by disulphide bonds to form the well-known Y shape of the antibody molecule
• our genome contains one heavy chaingene, and two light chain genes (x and lambda)
• but any individual B cell can only ever make an antibody using one heavy and one light chain
• so never a mix of x and lamda

Question 13

Describe the structure of immunoglobulin

Answer 13

• in the top part of the Y arms, each Ig has two variable regions
• one from the light chain and one from the heavy chain, called V_L and V_H, respectively
• these are at the N terminus of the Ig protein
• the variation in amino acids at the ends of the arms enables different Igs to bind to different targets
• this region is called the antigen-binding site
• beyond the variable Ig region is the constant region, called C_L and C_H
• the light chain has one Ig domain in its constant region
• but the heavy chain constant region has 3 or 4 constant domains, depending on the antibody class
• Three constant Ig domains (CH1- CH3) for IgG, IgA and IgD
• four (CH1- CH4) for IgE and IgM.

Question 14

What is similar and different between the BCR and antibody?

Answer 14

• the BCR and antibody share the same structural features in the immunoglobulin, but the BCR has an extra transmembrane region in its C terminus (green in image)
• this keeps the receptor anchored in the B cell’s plasma membrane
• the Ig domains are the extracellular part of the BCR

Question 15

Interpret this diagram to explain how the heavy chain gene is organised to allow both antibody and BCR molecules

Answer 15

• The schematic below shows how the heavy chain gene is organised to allow it to make both antibody and BCR molecules.
• Soluble (antibody) and membrane bound (BCR) Ig are transcribed from the same gene, but which mRNA is produced depends on alternative polyadenylation and splicing (which you discussed last year in MCB and this year in GEN).
• The transcript can be either polyadenylated early, to leave out exons encoding the trans-membrane domain (green), or spliced (and polyadenylated later) to omit the secretion signal (pink).
• Can you see how the primary transcript is rearranged – based on different sites of polyadenylation and splicing – to produce the mRNA encoding for either the secreted form or the membrane bound of the Ig?
• Since both transcripts come from the same Ig heavy chain gene, you can tell that for any single B cell the secreted and membrane bound form of Ig must have identical extracellular domains, and therefore bind to the same antigen!

Question 16

What are the three functional regions of Ig?

Answer 16

• Fab: two fragments corresponding to the antigen-binding regions, that are the two arms of the Y structure broken off the stem
• Fc (fragment crystallisable): one fragment corresponding to the stem of the Y, formed by the terminal part of the constant region

Question 17

Explain the function of the Fab domain

Answer 17

• the Fab domain of the antibody contains the variable region
• it is therefore part of the antibody which binds to the antigen

Question 18

Explain the function of the Fc region

Answer 18

• the Fc region is crucial for the effector functions of antibodies by binding to various antibody receptors (Fc receptors)
• this includes:
• opsonisation that allows immune cells to bind and act, such as phagocytosis or degranulation
• complement fixation (via C1q)
• mother-to-baby antibody transfer across the placenta and into breast milk

Question 19

What is the complementarity-determining region (CDR)?

Answer 19

• looking at the structure of the IgH variable domain, there are three loops in red that contact the antigen
• this is called the complementarity-determining region (CDR)
• CDR1 and CDR2 loops are the most genetically variable parts of the V segment, while CDR3 arises during VDJ recombination
• it is the diversity of sequence in these loops (and equivalent loops in the variable domain of the light chain) that allows our body’s collection of Ig molecules to bind specifically to such a wide variety of targets

Question 20

Which of the following describes the correct composition of human Ig?

Answer 20

• A heterotetramer of two identical light chains and two identical heavy chains all connected by disulphide bonds

Question 21

What are the functions of the constant domains of an Ig heavy chain? Select all that apply.

Answer 21

• opsonization
• transfer of Ig across epithelia, mediated by Fc receptors
• You may have realised that the constant domains are not all the same (different antibody classes have different C regions). However, the constant domains (through the Fc region) allow all antibodies (which have widely different specificities) to be treated equivalently by other elements of the immune system. This is why the constant region drives effector functions, whereas the V region drives specificity

Question 22

What are the five distinct major classes of Ig in most higher mammals?

Which of these have additional subclasses?

Answer 22

• IgG: additional subclasses
• IgA: additional subclasses
• IgM
• IgD
• IgE

Question 23

How do different classes of Ig differ and are similar?

Answer 23

• These differ in size, charge, carbohydrate content and the way they can assemble
• e.g. IgG, IgA and IgD have three constant doains each heavy chain
• IgE and IgM have four
• they all are the typical Ig structure
• even when a B cell changes the Ig class that it produces, the same V region is used, regardless of which antibody class the cell is producing

Question 24

Describe IgM

• structure
• function

Answer 24

• IgM is the first Ig to be expressed on B cells as they mature, so is the first to encounter an antigen.
• It makes up approximately 10% of the antibody in adult serum.
• Soluble IgM subunits form as a pentamer (recently discovered to be rotationally asymmetric - see the cryoEM image on right of the figure) connected by another polypeptide - the J chain.
• The pentamer is held together by disulphide bonds between cysteine residues.
• It therefore possesses 10 potential antigen binding sites providing this subclass with high avidity for antigens, even when the affinity of individual binding sites is low.

Question 25

What is avidity?

Answer 25

• Avidity is the overall strength of the binding of an antibody to an antigen with multiple binding sites.
• It is different from affinity which refers to the strength of binding at a single site.

Question 26

Describe IgG

• abundance
• subclasses
• function

Answer 26

• IgG is the most abundant Ig in normal human serum
• accounts for 70-75% of the total Ig serum pool of a healthy adult.
• In humans there are 4 subclasses, IgG1, IgG2, IgG3 and IgG4 which have different types of g heavy chains (γ1, γ2, γ3, γ4)
• It is the main antibody class for responding to small pathogens inside the body.

Question 27

Describe IgA

• abundance
• subclasses
• function

Answer 27

• IgA makes up 15-20% of serum Ig.
• There are 2 subclasses in humans: IgA1 and IgA2 (α1, α2).
• As with IgM, IgA can contain a J chain that facilitates dimerization.
• IgA dimers are the main Ig in mucosal secretions (e.g. saliva, genitourinary, respiratory or gastrointestinal tracts), so is particularly important against pathogens that infect these locations.

Question 28

Describe IgE

• abundance
• location
• function

Answer 28

• IgE makes up less than 1% of plasma Ig, but is found in abundance bound to the surface of basophils and mast cells in all humans.
• IgE is important in the response to parasitic helminth infections, but it also plays a role in allergic diseases such as asthma and hay fever, diseases that are more common in developed countries, where people are rarely exposed to parasites.

Question 29

Describe IgD

• abundance
• location
• function

Answer 29

• IgD makes up less than 1% of secreted plasma Ig.
• It is present in membrane bound form alongside IgM on the surface of naïve B cells (fully developed B cells that have never encountered an antigen that their Ig binds to).
• IgD is thought to play a role in the activation of B cells prior to their differentiation into antibody-secreting cells, but its function(s) remain(s) something of a mystery.

Question 30

What is the T cell receptor?

Answer 30

• Just like B cells, T cells have receptor molecules on their cell surface that are individual to that cell clone.
• The T cell receptor (TCR) is a heterodimeric transmembrane receptor, formed by two chains – TCR-α and TCR-β – connected by disulphide bonds.
• However, there is no secreted form of the TCR.

Question 31

Does the TCR operate alone?

Answer 31

• no, it exists as a complex with CD3 to form the TCR complex
• CD3 has an intracellular domain to which signalling molecules can bind, that allows the TCR to send signals into the cell when it binds to antigen, just like CD79 does for the BCR

Question 32

Describe the structure of the TCR

Answer 32

• both TCR chains contain a variable region (V) and constant region (C)
• Each is a single Ig domain.
• Both TCR chains have a C terminal transmembrane domain.
• Just like Ig, the variable region confers the specificity that allows different T cells to recognise the unique characteristics of different antigens: variations in the three CDR loops of the variable Ig domain define which antigen each TCR is able to bind.

Question 33

Is the alpha/beta TCR able to recognise and bind directly to a free antigen?

Answer 33

• The αβ TCR – unlike the BCR or soluble antibodies – is unable to recognise and bind directly to a free antigen.
• In fact, T cells need to be introduced to the antigen by other cell types which have previously come into contact with the antigen and are thus acquaintances already.
• This process is called antigen presentation.

Question 34

What is the major histocompatibility complex (MHC)?

Answer 34

• it is a protein complex present on the cell surface
• cells process potentially foreign material and mount that material onto the MHC
• the variable region of the TCR recognises the whole antigen-MHC complex

Question 35

Describe the two types of MHC

Answer 35

• MHC Class I:
• present in every nucleated cell type in vertebrates
• it present peptides that come from inside the cell, are during synthesis or degradation
• antigens form intracellular infection (especially viral infections), as well as peptides made during synthesis of cell proteins, are present on MHC Class I
• MHC Class II:
• present only in a subset of immune cells, particularly macrophages, dendritic cells (DCs) and B cells, which are APCs
• antigens come from proteins made outside the cell, typically from material (e.g. dead cells or opsonised pathogens) captured and degraded in phagosomes

Question 36

What upregulates MHC Class II?

On what surfaces?

Answer 36

• MHC Class II can also be up-regulated on the surface of other cell types.
• This up-regulation is induced by cytokines produced in response to danger signals (PAMPs, DAMPs).

Question 37

What are co-receptors?

Where are they expressed?

What are the two co-receptors of interest?

Answer 37

• T cells also have co-receptors that help tell the T cell whether its TCR is binding to MHC Class I or MHC Class II.
• These co-receptors are CD8 and CD4, respectively.
• Most TCR molecules can only bind to either MHC class I or class II
• Therefore which of CD4 or CD8 is expressed not only distinguishes T cells for their ability to recognise a particular type of MHC, but also defines the way T cells act upon recognition of their target on a cell.
• Therefore, CD4 and CD8 are important molecules for defining T cell function.

Question 38

What does TCR alongside CD4 recognise?

On what cells?

What happens after?

Answer 38

• TCR alongside CD4 (on CD4+ T cells) recognises antigen presented on MHC Class II molecules.
• Upon binding their TCR to the MHC Class II, CD4+ T cells will respond by sending activation signals to the antigen-presenting cell (APC) presenting the antigen.
• This prompts the antigen-presenting cell (APC) to get more serious about attacking the pathogen (or for B cells to help in the development and production of its Ig, as we will see later).
• This is why CD4+ T cells are also called helper T cells (TH) as they help the activity and/or development of the immune system.

Question 39

What does TCR alongside CD8 recognise?

On what cells?

What happens after?

Answer 39

• T cells expressing CD8 (or CD8+ T cells) are able to recognise antigens presented on MHC Class I molecules.
• Detecting foreign antigen that arises in the cell means that the cell may be a ‘Trojan horse’, either infected or mutated (potentially cancerous).
• Therefore, upon TCR binding to MHC Class I, CD8+ T cells act directly against these cells by killing them.
• This is why CD8+ T cells are also called cytotoxic T cells (TC, CTL or killer T cells) and are important in the response to viral infections and cancer.

Question 40

What are T cells initially derived from and where?

Where does it then move to?

Answer 40

• T cells are initially derived from haematopoietic stem cells resident in the bone marrow.
• Unlike B cells, however, T cells develop from lymphoid progenitors that are seeded in the thymus and develop first into thymocytes, before going on to eventually differentiate into CD4+ or CD8+ T cells that can move out of the thymus and disperse into the secondary lymphoid organs and circulatory and lymphatic systems of the body.

Question 41

What are the two stages to the generation of the diversity of Ig and TCR?

Answer 41

• diversification
• adaptation

Question 42

How is the initial diversity of our immune system achieved?

Answer 42

• it is achieved at the level of the DNA of the individual immune cells through a process of somatic recombination

Question 43

Describe somatic recombination

Answer 43

• The loci where the Ig light and heavy chains, and the TCR chains are encoded contains many many alternate versions of the DNA segments used to make up the variable domain of the Ig and TCR molecules.
• The DNA segments that make up the variable domain are termed the variable (V), joining (J) and (for the IgH and TCR-β chains) diversity (D) regions on their corresponding gene.
• The table below shows the number of V, D and J regions in the main Ig and TCR genes. Don’t worry about the exact numbers - just note the large numbers of V regions in all these genes:- this is because two of the CDR loops are entirely encoded by this region.

Question 44

Use this diagram of the genetic structure of the gene loci encoding the heavy and light chains of the B cell receptors to explain VDJ recombination

Answer 44

1. The heavy chain Immunoglobulin gene is found on chromosome 14 in humans.

While the lambda and kappa light chain genes are found on chromosome 2.

2. The heavy chain gene is comprised of what can be described as 4 distinct regions.
   - The variable region (often referred to as the V region), the diversity or D region, the joining or J region, and the constant or C region.
3. The variable, diversity and joining regions of the heavy chain are comprised of multiple distinct copies.
   - In humans there are 40 variable regions, 24 diversity regions and 6 joining regions.
   - In a final heavy chain gene only 1 of each is expressed.
4. The constant region encodes, as its name suggests, the constant domain of the heavy chain, and thus eventually will determine if the final immunoglobulin is an IgD, IgM, IgG, IgA or IgE.
5. Like the heavy chain, the light chain genes have variable and joining regions, but not diversity regions.
   - For instance in humans the kappa locus has 46 variable regions and 5 joining regions.

The key take home from this is that at germline the immunoglobulin genes provide options, through the process described in the video below the heavy chain of 40 V regions, 24 diversity regions and 6 joining regions are reduced to a single combination of one V, one D and J region, which can be different for each mature B cell.

Question 45

Does VDJ recombination affect both Ig and TCR?

Answer 45

• yes, both Ig and TCR are made from one gene that contains one each of the V, D and J regions, and one that contains one each of a V and J region.
• These are brought together by a series of random internal genetic rearrangements via a process called VDJ recombination.
• So, in humans, a mature B cell expressing surface Ig can have one heavy chain made up of any 1 of 40 V regions plus 1 of 24 D regions and 1 of 6 J regions, combined with either a kappa or lambda light chain made up of any 1 of 46 V regions and 1 of 5 J regions. Just imagine the possible number of permutations, or different antibodies or TCRs!

Question 46

How many possible combinations of V, D and J regions are there?

Why is this not enough and how is this solved?

Answer 46

• approx 2.5 million different Igs
• this may sound like a lot, but is much less than what would be necessary to guarantee that a host could respond to a given pathogen by generating a specific antibody capable of marking the pathogen for destruction, or disabling a critical component of its infectious life cycle in a reasonable amount of time, before the microbe had the chance to kill the host.
• Fortunately, the actual Ig repertoire of mammals, such as humans and mice, is thought to be ~1013 in size.

How does the immune system bridge this gap?

• First is the extra diversity introduced into CDR3 by the VDJ recombination mechanism

Question 47

Describe the VDJ recombination introduced into CDR3

Answer 47

• The immunoglobulin genes are composed of separate segments of DNA that become joined together by a process called somatic recombination to make a functional gene.
• In Heavy chain genes there are three gene segments: the variable or V gene segment, a diversity or D gene segment and a joining or J segment.
• Light chain genes such as those shown here have only two gene segments: the V and the J segments.
• Gene segments that can be recombined have specific sequence motifs adjacent to them t called recombination signal sequence, or RSS motifs.
• A protein complex containing the products of the recombination activator genes RAG1 and RAG2 bind specifically to the RSS motifs.
• In this example, flanking a V gene segment and a J gene segment.
• The individual gene segments to whose flanking RSS motifs the RAG protein complexes bind are selected at random from a number of copies located at each gene locus.
• The RAG protein complexes bring together the gene segments to be recombined and cleave the DNA exactly at the junction of the gene segment and its adjoining RSS motif.
• The cleavage creates a hairpin of DNA at the end of the gene segments and double-stranded breaks at the ends of the RSS motifs.
• Additional proteins – DNA-dependent protein kinase, Ku, Artemis, and a dimer of DNA ligase and XRCC4 – are incorporated into a large complex with the RAG proteins.
• These RSS ends are joined, forming what is called the signal joint, to create a closed circle of DNA that plays no further role in the recombination process.
• The DNA hairpins at the ends of the gene segments are then cleaved.
• An additional enzyme – terminal deoxynuclotidyl transferease or TdT – is recruited and adds additional nucleotides to the ends of the DNA strands.
• The other enzymes in the complex then ligate together the two ends of the gene segments completing the recombination process.

https://vimeo.com/298989012/1adcab733e?embedded=true&source=video_title&owner=71770559

Question 48

Considering the mechanism of VDJ recombination, make a note of which processes lead to additional diversity in the VDJ element

Answer 48

1. The addition of non-germline nucleotides by TdT will insert extra random bases;
2. Cleavage by Artemis leaving a 3’ overhang (i.e. cuts into the top strand of the hairpin) can lead to deletion of nucleotides from the end of the V, D or J region;
3. Cleavage by Artemis leaving a 5’ overhang (ie cuts the bottom strand of the hairpin, that can then open out) generates a nucleotide palindrome.

Question 49

How does allelic exclusion apply to making antibodies/BCR?

Answer 49

• remember that a process of allelic exclusion [where one allele is active while the other is silenced] also occurs,
• so that only one of the 2 chromosomal copies of each gene will be actively producing the final antibody, so each B cell can make only one antibody/BCR.

Question 50

How does the T cells receptor (TCR) generate its diversity?

Answer 50

• The T cell receptor (TCR) generates its diversity through VDJ or VJ recombination in essentially the same way as the immunoglobulin genes.
• The TCR genetic locus is shown below.
• Note that due to the layout of the TCRβ gene, the Cβ1 constant region can only be used with the D and J regions that precede it, and the Dβ2 region can only be joined to the J regions that follow it.

Question 51

Describe the TCRs of the unconventional T cell: gamma-delta T cells

Answer 51

• The γδ T cells use alternative genes for making their TCR: the alternative chains these genes encode are called TCR-γ and TCR-δ.
• The γδ TCR is unlike the classical αβ T cells in that it is activated in an MHC-independent manner - that is to say they bind to ‘free’ antigens rather than those presented on MHC, making their antigen recognition process conceptually more similar to the BCR than the αβ TCR.
• The γδ TCR genes are rearranged in much the same way as αβ TCR and Ig genes.
• However, these genes are much less diverse, having fewer V, D and J gene segments, as shown in the table below.
• The γδ T cells are most prevalent in the mucosa, and are thought to be important for recognising lipid antigens, but are also significant players in immunotherapy against cancer, (an area you will cover both in CBIO (if you are doing it) and in IMI10). They may also sometimes contribute to autoimmunity, which you will explore in IMI9.

Question 52

Describe the TCRs of the unconventional T cell: MAIT cells

Answer 52

• MAIT cells have an αβ TCR, but their TCR does not recognise antigen presented on MHC class I or MHC class II molecules.
• Rather they usually recognise antigens presented on an alternative MHC molecule, MR1, which is highly conserved in humans (unlike MHC, which is enormously diverse).
• The variable domains of MAIT cell TCRs usually arise from a very small selection of V D and J region combinations, that give the TCR specificity for MR1.
• Also, as a result of not recognising either MHC class I or MHC class II molecules, MAIT cells do not normally express either CD4 or CD8 molecules.
• The ligands (antigens) presented by MR1 are thought to be metabolites that are produced by a wide range of bacteria, but other ligands may exist.

Question 53

Describe the TCRs of the unconventional T cell: NKT cells

Answer 53

• The TCRs of NKT cells is conceptually similar to that of MAIT cells: it is an αβ TCR that recognises non-protein ligands presented by a non-conventional MHC molecule, and the TCR is usually made up of a specific subset of V, D and J segments.
• In this case, NKT cells TCR recognises glycolipids presented by the CD1d molecule.
• They are thought to be important against certain bacteria (eg mycobacterium Tuburculosis) but NKT cells are also implicated in autoimmunity.

Question 54

What are the main components of T cell receptor complex?

Answer 54

• TCR-α
• TCR-β
• CD3

Question 55

How is the complementarity-determining region (CDR), the already most diverse part of the Ig due to VDJ recombination, further diversified when B cells encounter antigens?

Answer 55

• the CDR is further diversified through a mechanism called somatic hypermutation (SHM)

Question 56

Describe how somatic hypermutation (SHM) works

Answer 56

• SHM introduces errors (mutations) into the CDR of the re-arranged Ig genes.
• SHM is driven by an enzyme called Activation-Induced cytidine Deaminase, or AID.
• AID causes an approximately million-fold increase in mutation rate of this region, compared to other parts of the genome.
• Mechanistically, AID catalyses the deamination of cytosine base to uracil in the DNA sequence.
• Because Uracil naturally base pairs to an A, this represents a C to T mutation in the Ig gene, (or G to A in the gene, if the C to T mutation is on the reverse strand of the DNA from the coding region).
• However, because uracil residues are not normally found in DNA, our genome repair processes may fix these ‘errors’, with uracil bases being removed and replaced with almost any other nucleotide(s).
• Because the process occurs while the B cell proliferates (because the B cell has been activated by the pathogen) the daughter cells of the mutating B cell can each have unique combinations of mutations slightly modifying their BCR, and potentially affecting the specificity and affinity of the BCR for the antigen.

Question 57

How is the process of somatic hypermutation triggered?

Answer 57

• The process of somatic hypermutation is triggered only after antigen-BCR binding in the secondary lymphoid organs (spleen, lymph nodes).
• It is in these secondary lymph nodes that most B cells hang out, after maturation (including VDJ recombination) in the bone marrow.
• It is here, in fact, that naïve B cells are thought to encounter many antigens brought by APCs, particularly dendritic cells
• When their BCR recognises an antigen, the B cell is activated and proliferate.
• This leads to the formation of germinal centres (GC), which are anatomically distinct regions within the B cell follicles of the lymphoid tissue (see figure below).
• Here, these activated B cells differentiate into so-called germinal centre B cells.
• It is in these cells that AID becomes active.
• They interact tightly with specialised stromal cells called follicular dendritic cells (FDCs) and CD4+ T cells, to regulate the process of SHM and the concurrent process of affinity maturation.

Question 58

What happens to B cells in different parts of the germinal centre?

Answer 58

• Dark zone:
• AID levels are high and your active Ig genes will undergo many mutations
• if your antibody cannot be made (e.g. frameshift or stop codon), you will die
• you may also be triggered to switch antibody class here e.g. IgM to IgG
• Light zone:
• this is where B cells take their new BCR to show to dendritic cells, compete for antigen and show off their processed antigen to T cells
• if they lose the fight to bind antigen, or the antigen they bind is not recognised by the T cell they die

Question 59

How does the body ensure that the new antibodies are better than the old ones?

Answer 59

• the germinal centre becomes a region of change and competition so that only the stickiest survive, in a process called affinity maturation
• Affinity maturation is the process whereby antibodies become better at their job (higher affinity) as the time goes on.
• The more a collection of BCRs see an antigen, the better they get at binding (up to a certain theoretical limit).
• This may be why some vaccines use booster to give you even better immunity than just one jab.
• It is due to the combination of somatic hypermutation and clonal selection.

Question 60

Describe antibody affinity maturation

Answer 60

• The encounter of a B cell with an antigen determines its maturation and selection.
• In this phase, a range of Ig produced by different B cells may have different affinities for the antigen, where affinity is defined as the strength of the binding of one antibody CDR to the antigen at a single site.
• Two binding processes are required for the B cell to survive in this environment.
• If either of the following signals is missing, the B cell will die:
  1. Its BCR must bind to the antigen provided by follicular dendritic cells (FDCs). FDCs are present only in a limited number in the germinal centre (GC). This creates a very competitive environment for the binding of the antigen, and only Ig with the highest affinity will be able to effectively bind.
  2. The B cell internalises BCR-bound antigen and presents it on MHC class II. The TCR of a helper T cell must bind to this antigen on MHC class II and receive help (more on this in IMI6) from the T cell.
• So B cells in the germinal centre (GC) that either do not win the competition to bind to antigens, or bind (and present on MHC class II) something that is not detected by CD4+ T cells* then the B cells undergo apoptosis.
• For instance: if the Ig binds a self antigen, then its presented peptides will not be recognised by the T helper cells, because T cells whose TCR binds self peptides are destroyed during T cell development in the thymus: more on avoiding self-recognition in IMI9
• The surviving cells (those that bind most strongly to a non-self antigen) keep proliferating and produce new variability by further cycles of SHM as the B cells divide and diversify in the dark zone, and test their affinity in the light zone.
• The process of SHM, coupled with the affinity maturation, can be understood as a micro-evolutionary process of survival of the fittest.

https://youtu.be/qGsyBwDVnTU

Question 61

What is class switch recombination (CSR)?

Answer 61

• As well as developing antibody with a high affinity, the immune system also needs to decide which isotype of antibody is required.
• This is also set in the germinal centre via class switch recombination (CSR).
• According to the signals received by the B cell, the heavy chain may change from one type to another, through genetic recombination.
• A naïve B cell (making IgM and IgD) might receive signals telling it to switch to one of the IgG isotypes (in the body) or an IgA (at mucosal surfaces), or IgE (against multicellular invaders).
• The heavy chain gene contains a sequence of constant regions encoding for the constant domains of the Ig classes.
• In humans this order is shown in blue in the figure below.
• Note that Cµ and Cδ are adjacent, as naive B cells make both IgM and IgD BCRs by alternative splicing.

Question 62

Describe how CSR works

Answer 62

• During CSR, a series of enzymes (including our old friend AID) introduce DNA double strand breaks (DSBs) and chop out the intervening DNA, containing unused constant regions and the remaining sequence is then re-joined.
• With the exception of IgD and IgM, only a single BCR class can be expressed at the same time.
• Take a look at the diagram below to see how this process occurs when class switching from IgM to IgG1.
• At the bottom of the panel you will notice that the segment order VDJ-γ1 sequence allows splicing of the mature transcript for the IgG1 heavy chain.
• Precisely how the immune system decides which isotope is needed remains something of a mystery.
• It is undoubtedly shaped by the combination of PAMPs and signals changing the cytokines released by the T cells and APCs (more of this in IMI5), but the details are poorly understood.

Question 63

How are plasma cells formed?

Answer 63

• At each stage of the process of affinity maturation and class switch recombination, some high quality B cells will be directed to differentiate into plasma cells.
• These are terminally differentiated mature B cells that produce the soluble form of BCR, the amazing antibodies.
• The plasma cells will migrate to the site of infection, or secrete their antibodies into the body fluids - blood and lymph.

Question 64

How are memory B cells formed?

Answer 64

• As antigen in the germinal centre runs out (because the infection has been defeated), a small proportion of the B cells with these high affinity antibodies return to a resting state similar to naïve B cells.
• These persist in a relatively quiescent form as memory B cells.
• In the event of a re-infection, these cells can quickly proliferate and differentiate to produce more plasma cells that rapidly produce antibodies to fight a second infection of the same (or similar) kind.
• This is one of the principles behind vaccination and you will learn more about immunological memory in IMI8.

Question 65

Match the letter from the image to the process (captial) or effector (lowercase)

Answer 65

Question 66

Does TCR diversity come from somatic hypermutation?

Answer 66

• no, TCR diversity only comes from VDJ recombination
• TCR genes cannot undergo further diversification

Question 67

What is the magnitude of TCR repertoire and antibodies?

How come?

Answer 67

• While it is difficult to determine the magnitude of the T cell receptor (TCR) repertoire, estimations based on next generation sequencing (NGS) suggest that the number of different T cell receptors (TCRs) in the body is around 106 (its variation comes only from VDJ recombination), compared to the potentially 1013 range for antibodies (which gain extra diversity from somatic hypermutation).
• The lower diversity is also a reflection of the fact that TCRs recognise antigen as linear peptides in MHC, whereas antibodies often recognise the 3D structure of a protein and not just its primary sequence

Question 68

If the TCR does not modify its receptors to adapt to pathogens, how does it adapt to antigens?

Answer 68

• through priming and anergy
• Rather than modifying their receptor, T cells adapt to antigen encounters by boosting the numbers and functions of T cells with desirable specificities, and suppressing T cells whose specificity is undesirable (eg against harmless self or environmental peptides).
• So once a T cells has responded to an antigen through its TCR (if associated with the correct additional signals - see IMI5) it will be activated, and proliferate to become more numerous.
• When the stimulation has left, the T cell will return to a ‘primed’ state: one that is much more quick to activate (i.e. the T cell is ‘primed’ to react quickly when it encounters its antigen).
• The long lived version of these primed T cells are an example of the memory cells, which we will cover more in IMI8.
• Conversely, if a TCR binds to antigen in the absence of additional danger signals, then the cell will take that as an indication that the antigen it senses is harmless.
• This will result in the T cell shutting down into a dormant state from which it is much harder to reactivate: a state of anergy.
• This helps us to avoid responding inappropriately to self-antigens, or to harmless external antigens.

Question 69

List the types of cells expressing MHC Class I and MHC Class II

Answer 69

• MHC Class I is expressed in every type of nucleate cell in vertebrates
• MHC Class II is expressed by antigen presenting cells including macrophages, dendritic cells and B cells

Question 70

What are the two forms of B cell immunoglobulin?

Answer 70

• either membrane bound (B cell receptor)
• or secreted (antibody)

Question 71

How does antibody diversity/specificity arise?

Answer 71

• Antibody (Ab) diversity/specificity arises:
• first in isolation [VDJ recombination]
• then in response to specific antigen (Ag)
• [somatic hypermutation (SHM) + clonal selection = affinity maturation]

Question 72

What does B cell survival and differetiation in germinal centre require?

Answer 72

1. BCR binding to opsonised antigens held on the surface of follicular dendritic cells (fDCs)
2. TCR of follicular T cell binding to peptides presented by B cell’s MHC Class II
3. Cytokine secretion driven by PAMP recognition and signals 1 and 2

Question 73

Briefly describe what types of antibodies are produced after class switching

Answer 73

• IgM: multivalent (repetitive surfaces)
• IgG: circulation, long-lived
• IgA: mucous surfaces
• IgE: granulocyte activation

Question 74

Observe the phases of B cell development

Answer 74