Case 17- Immunity 2

Question 1

The 5 different isotypes of immunoglobulins

Answer 1

IgM, IgD, IgG, IgA and IgE

Question 2

How the heavy chain determines the immunoglobulin class

Answer 2

The constant region of the heavy chain can consist of either γ (gamma; IgG), α (alpha; IgA), μ (mu; IgM), ε (epsilon; IgE) or δ (delta; IgD) domains. IgG, IgA and IgD have 3 constant domains with a hinge region, whereas IgM and IgE have 4 constant domains but no hinge region.

Question 3

Structure of the different antibodies

Answer 3

IgG, IgE and IgD are monomeric (1 subunit), secreted IgA is usually present as a dimer (2 subunits; but can be secreted as a monomer) and secreted IgM exists as a pentamer (5 subunits).

Question 4

IgG

Answer 4

IgG in humans is found as 4 classes: IgG1, IgG2, IgG3 and IgG4, named in order according to their decreasing abundance in the serum. IgG isotypes produced during an immune response can be found in the bloodstream and extracellular spaces in tissues. Has 1 subunit.

Question 5

IgA

Answer 5

IgA antibodies are found as 2 subclasses: IgA1 and IgA2. IgA can be found in the bloodstream but also acts in defence of the mucosal surfaces- it is secreted into the gut and respiratory tract, as well as the mother’s milk. Has 2 subunits

Question 6

IgM

Answer 6

Secreted IgM antibodies have a high molecular weight as they exist as pentamers, due to their size they are normally present in blood but not tissues. Has 5 subunits

Question 7

IgD

Answer 7

The monomeric form of IgM along with IgD exists on the surface of a naïve B cell (membrane), known as B cell receptors (BCR). The first antibodies to react to a new antigen whilst class switching i.e. changing to another form of the antibody whilst retaining antigen specificity. Has 1 subunit.

Question 8

IgE

Answer 8

IgE is particularly involved in defence against multicellular parasites i.e. schistosomes but also involved in common allergic diseases i.e. asthma. Has 1 subunit

Question 9

The different functions of an antibody?

Answer 9

Opsonisation- phagocytic cells have antibody (FC) receptors
Agglutination- antigen and antibody (IgG or IgM) clump together because immunoglobulins can bind to more then one epitope simultaneously
Neutralisation- binds to pathogens preventing their attachment to cells
Antibody dependent cell mediated cytotoxicity- the antibody-antigen complex can bind to cytotoxic cells i.e. cytotoxic T cells via the Fc component of the antibody, targeting the antigen for destruction
Complement activation- IgG or IgM can activate the classical pathway and IgA activates the alternate pathway
Mast cell degranulation- cross linkage of IgE bound to mast cells and basophils results in degranulation
Protection of the neonate- transplacental passage of IgG and secretion of IgA in breast milk protects the newborne

Question 10

Aims of B cell development

Answer 10

• Generate diverse antigen receptors (VDJ recombination, junctional diversity)
• Eliminate self reactive B cells/receptors (B cell tolerance)
• Mature activated B cells to make a better antigen fit (somatic hypermutation and affinity maturation)
• Choose the best class of antibodies for the job (isotype class switching)

Question 11

Antibody diversity

Answer 11

Antigenic receptors are created through genetic recombination, where segments of information are cut and pasted from the gene. Rearrangement of gene segments allows antibodies with a variety of specificity to be produced from a relatively small amount of DNA.

Question 12

How is antibody diversity generated

Answer 12

1) Combinatorial diversity- genetic rearrangement
2) Junctional diversity
3) Affinity maturation
4) Class switching

Question 13

Where does combinatoral diversity- genetic rearrangement take place

Answer 13

Before genetic rearrangement occurs, gene segments are in a germline configuration. The rearrangement only ours in the variable domain

Question 14

Combinatorial diversity- genetic rearrangement B cells

Answer 14

1) Each variable domain is encoded by a random combination of one of each of the V (variable), D (diversity heavy chain only) and J (joining) exons (nucleic acid sequence in the gene) from each gene complex.
2) There are multiple different copies of each gene segment (V, D, J). Each chain will select one of each V,D and J gene.
3) These genes are picked out through VDJ recombination. The heavy and light chains have a constant (FC region) which joins to the cell. The heavy chain has a VDJ segment and the light chain only has a VJ region.
4) The different exons give rise to thousands of possible genetic combinations for both heavy and light chains.
5) Heavy chain genes are rearranged before light chains.

Question 15

Step 1 of Combinatorial diversity- genetic rearrangement

Answer 15

A random D gene segment is joined to a random J gene segment by looping out of the excess DNA. This process is catalysed by RAG1 and RAG2 proteins. These RAG proteins are ‘DNA shufflers’ and are only expressed in developing lymphocytes whilst they are assembling their antigen receptors. Deficiency of these RAG enzymes can lead to severe combined immunodeficiency disorder (SCID).

Question 16

Step 2 of combinatorial diversity- genetic rearrangement

Answer 16

RAG proteins also join a random V gene segments to the DJ gene segment. The DNA between the D and V segment are removed. This creates one continuous exon which will code for the variable region of a heavy chain. There is repair of the double stranded break.

Question 17

Step 3 of Combinatorial diversity- genetic rearrangement

Answer 17

1) C exons encode the constant regions. Heavy chain C segments are clusters of exons, each of which encode either a domanant or hinge region of the constant region.
2) Heavy chain DNA contains 9 constant regions corresponding to the 5 different immunoglobulin classes and their various subclasses Cμ, Cδ, Cγ3, C γ1, C α1, Cγ2, Cγ4, Cε and C α2.
3) In an unstimulated B cell, the heavy chain mRNA that is transcribed contains both the Cμ and Cδ segments. One or more of these constant regions is then joined to the combined VDJ segment on the mRNA in a process called ‘differential splicing.’ The mRNA can then be translated into a functional heavy chain. Heavy chain rearrangement occurs before light chain.

Question 18

The difference and similarities between the production of the light versus heavy chain

Answer 18

• Light chain V region genes are constructed from two segments- a variable (V) and a joining (J) segment are joined to form a complete light-chain V region exon. The light chain C region is then joined to the V region.
• Heavy chain V regions are constructed from three gene segments- the diversity (D) and J gene segments join, then the V gene segment joins to the combined DJ sequence, forming a complete V exon. A heavy chain C region gene is encoded by several exons.

Question 19

Combinatorial diversity- T cells

Answer 19

Instead of heavy and light chains its beta and alpha, beta produces a VDJ segment and alpha produces a VD segment

Question 20

Junctional diversity

Answer 20

The formation of the junctions between various gene segments provides increased diversity, where nucleotides can be added or removed to form the joining segment:

1. Junctional flexibility- slight variations in the position of segmental joining when exons are spliced (joined together)
2. Nucleotide addition- nucleotides can be added to the V-J, V-DJ or VD-J joins by the enzyme TdT (terminal deoxynucleotidyl transferase)
3. Nucleotide deletion

Question 21

Problems with Junctional diversity

Answer 21

This process leads to non-functioning cells in 2/3 of cases. B cell progenitors with non-functional immunoglobulins will never become mature B cells and hence this diversity is only achieved at the expense of considerable loss of cells during development.

Question 22

Diseases due to mutations in B cell development

Answer 22

• Mutations in RAG- SCID (severe combined immunodeficiency) syndrome
• Mutations in double stranded DNA repair proteins- SCID, developmental problems, cancer, neurodegenerative diseases
• Both will require a Bone marrow (stem cell) transplant

Question 23

B cell tolerance

Answer 23

They get shown the antigens inside bone marrow, if they react to any of those self antigens they get to rearrange their light chain gene and come up with new receptors which don’t bind to self antigens (receptor editing). The bone marrow contains the same antigens as in the secondary lymphoid organs in which naïve B cells circulate.

Question 24

Activation of B cells- need to be exposed to an antigen or T cells

Answer 24

• In the lymph node there is a T cell and B cell area
• Humoral antigens are tagged by compliment and go to the B cell area
• Antigens in the blood are tagged by dendritic cells and go to the T cell area.
• Within the B cell area are lymphoid follicles which are areas made up of follicular dendritic cells and B cells.
• Follicular dendritic cells displays antigens to B cells within the lymphoid follicules
• In the T cell area, the T cells will be activated by a dendritic cell displaying an antigen. The corresponding B and T cell will interact together and the T cell gave a second signal to the B cell to begin proliferation
• The lymphoid follicle is where B cell proliferation takes place, now called a germinal centre
• Replication introduces small errors in the B cell receptor, these make it either a worse fit or an even fit for the antigen.

Question 25

Class switching

Answer 25

The first immunoglobulin expressed by a B cell is IgM or IgD. However, in the immune response the same variable region can be expressed with a different constant region. The process of changing the constant region is known as class switching. A single B cell can produce different classes of immunoglobulins that are specific to the same antigen.

Question 25

Affinity maturation

Answer 25

1) The process that makes an antibody a better fit for its antigen.
2) When activated B cells are replicating to increase in number in order to combat an infection, they express an enzyme called AID (activation induced cytidine deaminase) which creates mutations in the variable domain. Known as Somatic hypermutation.
3) Mutation in cells is normally tightly regulated to avoid cancer formation.
4) Antibodies with mutations that result in a higher affinity for an antigen are positively selected for. However, some mutations can decrease an antibody’s specificity for the antigen. These are destroyed by apoptosis.

Question 26

Process of class switching

Answer 26

1) Before each heavy chain C gene segment (except Cδ) there is a DNA sequence called a switch site.
2) During the course of an immune response, an enzyme called AID (activation-induced cytidine deaminase) initiates class switching. This is critical for the class switch from IgM in the primary response to IgG, IgA or IgE in the secondary response.
3) During switch recombination, two switch sites are joined together and in this process heavy chain C gene segments are looped out and deleted. The switch site adjacent to the μ constant region can be joined to any of the other switch sites to produce the different immunoglobulin classes.
4) For example, the switch site adjacent to the μ constant region joins to the switch site adjacent to the ε constant region to produce an IgE expressing cell.
5) Cytokines are important for controlling which class is chosen.

Question 27

What are B and T cell receptors specific for

Answer 27

1antigen

Question 28

The B cell receptor

Answer 28

The B cell receptor is a membrane bound immunoglobulin with the Y shaped antibody structure which is anchored to the membrane by 2 transmembrane immunoglobulins Igβ and Igα. The receptor is highly specific and binds to antigens leading to the activation of B cells via thymus-independent antigen activation.

Question 29

B cell rearrangement- same as antibody gene rearangement

Answer 29

• Heavy chain genes are rearranged with the joining of the D and J heavy gene segment. It’s the joining of the V heavy chain to the new DJ heavy locus. Forms the heavy chain VDJ rearrangement and leads to the expression of the heavy chain.
• Light chain genes begin to rearrange with V and J joining. If these genes fail to produce a functional light chain, the rearrangement occurs at different gene segments.
• Upon formation of a functional light chain, IgM immunoglobulin is expressed on the surface of a B cell

Question 30

B cell development overview

Answer 30

1. B cells derive from haematopoietic stem cells located in the bone marrow.
2. B cell progenitors remain in the bone marrow to undergo maturation. Bone marrow stromal cells create a microenvironment where B cell progenitors are committed to becoming B cells.
3. Pro-B cells undergo rearrangement of the heavy chain locus (VDJ) catalyzed by RAG.
4. A pre-B cell receptor is made consisting of the heavy chain locus and a surrogate light chain.
5. This causes RAG to be re-expressed and catalyze the rearrangement of the light chain locus.
6. Upon successful formation of a light locus, an immature B cell is formed that expresses IgM on its surface. Here the immature B cell is tested for auto-reactivity, if it is it will undergo apoptosis.
7. This immature B cell migrates to the spleen and undergoes alternative splicing that leads to the expression of IgD as well as IgM.
8. In the spleen, the B cell requires many positive signals, such as cytokines and BAFF (B cell activating factor) expressed by follicular dendritic cells, to fully mature.

Question 31

T cell receptor (TCR)

Answer 31

The T cell receptor consists of an alpha and beta domain with CD3 structures on either side of the receptor to help activate T cells

Question 32

T cell receptor gene rearrangement

Answer 32

• Beta gene rearrangement- In the formation of TCR, β gene segments rearrange first. Beginning with D joining to J and subsequently V joins to DJ. Cells then undergo proliferation.
• Alpha gene rearrangement- Proliferation comes to a halt and the cell can undergo α gene rearrangement. α genes only consist of V and J segments, however, several cycles of rearrangement can occur until a useful α chain is produced.
• TCR formation- the alpha chain binds to the beta chain on the cells surface with CD3 and the cell can undergo selection.

Question 33

TCR variation

Answer 33

The γ:δ receptor recognises antigens directly and are rapid responders (don’t need MHC molecules). γ:δ T cells also undergo gene rearrangement but there are fewer gene segments so variability is limited.

Question 34

T cell development overview

Answer 34

1. T cells derive from haematopoietic stem cells in the bone marrow.
2. T cell progenitors leave the bone marrow and migrate to the thymus for development and maturation
3. When T cell progenitors enter the thymus they are ‘double negative’ - negative for CD4 and CD8, CD3 and the T cell receptor
4. Thymic stromal cells then commit T cell progenitors to the T cell lineage via a signalling receptor (Notch-1) that initiates T cell receptor (TCR) gene rearrangement
5. The T cell begins to express both CD4 and CD8. The T cell is now ‘Double positive’
6. Genes for the β chain of the TCR rearrange first using V(D)J recombination. Unsuccessful rearrangement leads to apoptosis. The T cell also begins to express CD3. The α chain then also rearranges.
7. Positive selection: T cells are destroyed if TCR cannot recognise self MHC
8. Negative selection: T cells are destroyed if they react to self-antigen (but a small number of self-reactive T cells become T regulatory cells).
9. Single positive thymocyte formation: T cells stop expressing either CD4 or CD8.
10. Mature CD4 or CD8 T cells leave the thymus to undergo activation in secondary lymphoid tissues

Question 35

How can B cells be activated

Answer 35

Part of humoral immunity. The activation of B cells requires signalling from the B cell receptor and either a thymus dependent antigen or thymus independent antigen

Question 36

Thymus dependent antigen activation

Answer 36

1. The B cell presents an antigen on a MHC class II molecule that is recognised by a specific TCR on a TFH cell. Once activated the TFH cell expresses CD40L and secretes cytokines
2. CD40L on the TFH cell interacts with CD40 on the B cell. IL-21 and IL-4 cytokines secreted by the TFH cell interact with their specific receptors on the B cell to provide survival signals. Further cytokine production by the TFH cells can determine the type of antibody produced by the B cell
3. The B cell becomes activated, undergoes proliferation and differentiates into plasma cells

Question 37

Germinal centre

Answer 37

Germinal centres are in secondary lymphoid tissues and are the sites where B cells undergo proliferation, differentiation, affinity maturation and class switching. Its where B cells differentiate into plasma cells.

Question 38

Seperation of B and T cells

Answer 38

1) B and T cells are normally kept within different areas of the lymphoid tissue
2) Due to this physical separation and the diversity of B and T cell receptors, it is unlikely that a B and T cell recognising the same antigen will encounter each other
3) Chemokines secreted by stromal and follicular dendritic cells cause migration of B and T cells to areas where they can interact

Question 39

Thymus independent antigen activation

Answer 39

B cells are activated without help from TFH cells. Other surface receptors i.e. toll like receptors recognise ligands such as bacterial lipopolysaccharide and provide the second signal. The signal is strengthened through crosslinking of the BCR and toll like receptor. Pro-survival signals are then induced. Somatic hypermutation and class switching is dependent on helper T cells so antibodies which are thymus independent have a lower affinity and are less functionally versatile

Question 40

Key facts about Thymus dependent B cell activation

Answer 40

Co-stimulation= TCR on Tfh cells
Affinity- high
Strength of immune response- strong
Isotype switching- yes
Example of antigen- Diptheria toxin

Question 41

Key facts about Thymus independent B cell activation

Answer 41

Co-stimulation= other cell surface receptors i.e. toll like receptors
Affinity- low
Strength of immune response- weak
Isotype switching- No (only IgM)
Example of antigen- Bacterial lipopolysaccharide

Question 42

Immunological memory

Answer 42

Allows the immune system to respond more rapidly and effectively to pathogens. B and T cells can become memory cells. Memory responses are called secondary immune responses, tertiary immune responses and so on depending on the number of exposures to the antigen.

Question 43

B and T memmory cells

Answer 43

• B cell response: Antibodies made during secondary and subsequent responses exhibit different characteristics, e.g., higher affinity antibodies to antigen via somatic hypermutation and different antibody types via class switching
• T cell response: Memory T cells have enhanced trafficking patterns and effector functions compared to naïve T cells
During the secondary response reactivation of memory B cells is dependent on its interaction with its corresponding memory T helper cells

Question 44

Generation of immunological memmory- Naive phase

Answer 44

Before an antigen is encountered, there is a low number of antigen specific T cells and B cells and they are not physically close to secondary lymphoid organs

Question 45

Generation of immunological memmory- Primary response

Answer 45

When an antigen is encountered, antigen-specific naive T cells in the T cell zone are primed by dendritic cells and then migrate towards the B cell follicles. Antigen-specific B cells that encounter antigen also migrate to the border of the T cell zone, which allows the B and T cells to physically interact. Activated B cells then either differentiate into short-lived plasma cells or participate in the germinal centre reaction with help from T follicular helper cells (labelled TFH cells). Low levels of antigen-specific IgG production are detected around 1 week after antigen challenge.

Question 46

Generation of immunological memmory- Memory phase

Answer 46

After antigen is cleared, antigen-specific memory T and B cells are generated. Some of the memory B cell remain close to the germinal centre. Memory T cells derived from effector TFH cells are localised at the B cell- T cell borders or in B cell follicles so the memory cells are close together.

Question 47

Generation of immunological memmory- secondary response

Answer 47

Both B and T cell types are present in secondary lymphatic tissues so when the same antigen is encountered they can interact quickly and activate each other. Memory B cells work as antigen presenting cells to activate memory TFH cells. The reactivated T helper cells also activates the memory B cells which differentiate into plasma cells and secondary germinal centres form. Antigen specific IgG antibodies are produced

Question 48

Primary antibody response

Answer 48

Takes 4-7 days, antibodies generated are usually IgM with some IgG. Antibodies generated have a low affinity for the antigen

Question 49

Secondary antibody response

Answer 49

The immunogen must be a protein to initiate a secondary response. The response generated is larger. There is increased production pf IgG antibodies with some IgA or IgE production as appropriate. Antibodies have a higher affinity for the antigen then in the primary response.