lecture 1

Question 1

from where do your white blood cells and erythrocytes arise?

Answer 1

A single stem cell: Long Term-HSC (haematopoetic stem cell)
Each of us has a population of haematopoetic/blood producing stem cells which sit, as adults, in our bone marrow.
Occasionally one will develop into a Short Term -HSC and that cell is active in generating all of the white blood cell types that are in your body.
In order to produce this variety of WBCs, the individual HSC has to undergo a number of branching points and decisions.
Every day you are making millions of WBCs… a continuous process that goes on throughout your life.

LT-HSC > ST-HSC > LMPP (Lymphoid-primed Multipotent Progenitor) > ELP (Early Lymphoid Progenitor) > T cell/NK cell/B cell

Question 2

Where does immune cell development occur?

Answer 2

adult bone marrow

Question 3

What selects a B-lymphocyte?

Answer 3

The antigen

Question 4

What recognises the antigen?

Answer 4

Surface bound immunoglobulin/B cell antigen receptor. Once recognised the Ig will change form so that it can be released from the cell in a soluble form to move through the bloodstream.

Question 5

What is Burnet’s theory of Clonal Selection?

Answer 5

The idea that one B-cell is selected out of billions of B-cells. This cell then differentiates and proliferates.

Question 6

What happens to a B-cell that is not recognised by an antigen?

Answer 6

B-cells are quiescent unless stimulated to respond, so the B-cell will do nothing and eventually die to be replaced by a new cell if not recognised by an antigen.

Question 7

In what way are B cells different?

Answer 7

Superficially all B-cells appear the same. The major difference is the composition of its receptor, which is unique to each B cell. On a single B-cell all the receptors are the same.

Question 8

How many antigens are B cells capable of recognising?

Answer 8

As a population, they are capable of recognising every possible antigen in the universe, as individuals they are capable of recognising only one.

Question 9

Do B cells act on their own?

Answer 9

No - they often need instructions from CD4 T cells to proliferate and differentiate. This is a safety mechanism to ensure that the immune system does not just fire off at the earliest possible option because they do create a lot of damage.

Question 10

How does B cell recognition of antigen change with time?

Answer 10

The B cell introduces mutations into the binding sites thus creating antibodies with stronger affinity for the antigens over time. This is one of the B cell’s most dangerous features.
The nature of the antibody will also change.

Question 11

What are possible reasons for a failure to respond to an infection?

Answer 11

• No B-lymphocytes
• No CD4 T-lymphocytes
• No B or T lymphocytes
• No signal from the CD4 to the B cell
• Failure of the B cell to respond to the T cell signal

Question 12

What are primary immune deficiencies?

Answer 12

There are 8 classes of PIDs defined by the IUIS spanning the adaptive and innate immune systems.
PID are a large group of disorders (>200) that result in recurrent infections and that are NOT caused by other diseases, treatments, or environmental exposure to toxins.
Mostly genetic disorders and most are diagnosed in children under 1 year.

Question 13

What are examples of Combined T and B cell deficiencies?

Answer 13

• Severe combined immunodeficiency disorder (SCID)
• Complete DiGeorge syndrome
• CD40 and CD40L deficiencies (HIGM)

Question 14

What are examples of Antibody deficiencies?

Answer 14

• CD40 and CD40L deficiencies (HIGM)
• Ig deficiencies (X-linked agammaglobulinaemia, XLA; X-linked lymphoproliferative disease, XLP, selective IgA deficiency)
• Common Variable Immunodeficiency (CVID)

Question 15

Why do some of these deficiencies appear in more than one category?

Answer 15

Defects in some molecules principally effect antibody formation, but they have secondary effects in other systems. (CD40 and CD40L deficiencies) : appear in both T and B deficiences, and antibody deficiencies.

Question 16

What is a combined immune deficiency?

Answer 16

(= severe combined immune deficiency)

• refers to the combined loss of humoral and cellular immunity
• humoral means liquid immunity = antibody = B cell
• Cellular means cell-mediated = T cell
• Because CD4 T cells regulate B cells, defects in T cells only can be SCID

Question 17

What is an antibody deficiency?

Answer 17

• Refers to the loss of some or all humoral immunity with cellular immunity intact
• Absent B cells (but T cells are normal)
• Common variable immune deficiency (CVID)
• Hyper-IgM syndromes (HIGM)

Question 18

What is the structure of an antibody?

Answer 18

Antibody is a disulphide linked dimer of Heavy and Light chain heterodimers.
2 identical heavy chains + 2 identical light chains.
There are 9 different heavy chain classes: µ, ∂, gamma1, gamma2, gamma3, gamma4, alpha1, alpha2, and epsilon.
There are 2 different light chain classes, kappa, and lambda.
Both H and L chains contain constant (C) and variable (V) regions.
V regions differ between all Ig molecules, C regions are identical within a class of Ig.

Question 19

How are humans able to detect different scents? How does this relate to antibodies?

Answer 19

Humans have approximately 400 functional genes coding for olfactory receptors (another 600 pseudogenes). These each code for individual neurons that are able to detect one scent.
Since the human genome contains ~30,000 genes this means approximately 1/30 of our genes encode for odorant receptors.
Our B cells need to be able to detect significantly greater numbers of antigens. It would be impossible/a waste to have a gene coding for each individual antibody - there must be a different mechanism.

Question 20

How are Ig V gene segments formed?

Answer 20

By gene rearrangement:
Heavy chain: V (45 segments), D (23), J (6)
1. a random D element is joined to a random J - the things inbetween are lost
2. Random V element is joined to the DJ element - things in between lost
3. this now encodes a full polypeptide for the variable region and will be spliced onto the constant region to form the heavy chain

Light chain: V(35), J(5) - simpler

Question 21

Why use gene rearrangement?

Answer 21

If you look at the maths this method provides approximately 1,100,00 possible Vh/Vl combinations from 115 minigene segments. Human 2nd Ig light chain locus (lambda) is as diverse as kappa adding another 1,100,00 possible Vh/Vl combinations.
Actually even more diverse than this.
This shows an incredible amount of diversity from only a small amount of genome.

Question 22

How is diversity generated through gene rearrangement?

Answer 22

1. Random selection of minigene segments for joining at each locus
2. independent rearrangement at H and L chain loci
3. Imprecision of junctions

Question 23

How are genes rearranged in B (and T) Cells but not in other cells in the body?

Answer 23

• each gene segment is flanked by a ‘recombination signal sequence’
• These indicate to particular enzymes (RAG1, RAG2, HMG1) that they need to be cut and joined. These enzymes are unique to B-cells and therefore cutting at the RSS will only occur in B-cells. The enzymes don’t care what is next to this sequence - they will cut wherever it occurs.
• the recombination signal sequence is actually quite a long sequence therefore making it unique - the RAG genes will not cut random places in the genome
• The Rag enzymes do not cut neatly - there is some imprecision as to where this cut occurs,
• The enzyme stays bound to the segment of gene they have cut out (e.g. between the V and J segments)
• The exposed ends of the DNA fuse together to form hairpin ends
• From this point onwards the joining together of the two DNA pieces is done by enzymes that appear in every cell: every cell needs to repair DNA from random breaks etc e.g. DNA-PKcs, Artemis, Ku70, Ku80
• These enzymes reopen the hairpin ends and then the ‘repair’ of the double strand break is done by ligases e.g. XRCCA and DNA ligase IV
• TdT (Terminal deoxynucleotidyl transferase) works exclusively in heavy chain rearrangement by adding random nucleotides onto broken ends (also unique to lymphocytes)
• It is therefore the expression of RAG1/2 that determines whether the antibody genes will be rearranged: if you express it in a muscle cell, that cell will begin rearranging these genes

Question 24

What is RSS?

Answer 24

Recombination Signal Sequence: a unique nucleotide sequence that is the RAG recognition sequence, adjacent to each mini-gene segment

Question 25

What are RAG1/2?

Answer 25

Recombination Activating Genes: essential to Ig gene recombination, they provide the recognition and cleavage activity

Question 26

What is HMG1?

Answer 26

High Mobility Group 1 protein: chromatin binding, structural protein also required for rearrangement but not unique to Ig gene rearrangement

Question 27

What is the role of Artemis/DNA-Protein Kinase catalytic subunit (DNA-PKcs)/Ku70;80?

Answer 27

Recognition and synapsis of the DNA ends

Question 28

What is the role of DNA ligase IV/X-ray repair complementing Chinese hamster cells 4 (XRCC4)?

Answer 28

Joining DNA ends

Question 29

What is TdT?

Answer 29

Terminal deoxynucleotidyl transferase is unique to B cells and adds extra, random nucleotides to the broken ends at V-D.J junctions.

Question 30

How does the B cell integrate Ig gene rearrangment with its development and why?

Answer 30

- allows selection of productive arrangments Pre Pro-B: earliest stage, undergoes D to J joining at H chain locus Pro-B: attempts V to DJ joining. 1/3 attempts will be successful due to necessity to join in correct reading frame. If successful, the cell progresses to Pre-B, if unsuccessful on first allele it will try again but failure to successfully rearrange one H allele results in cell death. Pre-B: attempts V to J joining at light chain locus, kappa first then lambda (failure at all four alleles will results in cell death) (again only 1/3 will be successful) Pre-BII small: success at one allele of H and one of L results in complete Ig This progresses on to be an immature IgM+ B cell Incredibly wasteful process but the diversity provided by having mini-gene segments far outweighs the cost in terms of energy

Question 31

What sort of mutations block B Cell development?

Answer 31

- progress through B cell development is mediated by successful Ig gene rearrangement - immature B cells are only able to leave the bone marrow if they express a functional Ig on their surface - surface expressed Ig is called the B cell receptor (BCR) - However many mutations that nlock development are signalling not rearrangement proteins - Igalpha/beta are scaffolding proteins (block progress from Pro-B to Pre-B large), BLNK is an adaptor protein (blocks progress from Pre-B large to small), SYK (Pro-B to Pre-B) and BTK (Large to Small) are kinases, PAX5 (Large to small) and E2A (ELP to Pre-Pro-B) are transcription factors that specify the B cell gene expression programme. (SLC also blocks between large and small and RAG between Pro-B and Pre-B)