lecture 2

Question 1

How does the cell signal success in immunoglobulin VH gene rearrangement?

Answer 1

• The Ig heavy chain pairs with the two surrogate light chains (SLC)
• the SLC are the same in every B cell: they are not generated by rearrangement and serve the purpose of a generic light chain
• The IgH-SLC complex pairs with two signaling chains called Igalpha and Igbeta to form the pre-B cell receptor (pre-BCR)
• the pre-BCR sends the ‘success’ signal into the cell: this is reliant on the other molecules as opposed to the IgH-SLC complex which is incapable of signalling success on its own

Question 2

How does the cell signal success in Ig VL gene rearrangement?

Answer 2

• The Ig light chain pairs with the existing Ig heavy chain to form a complete Ig
• The IgHL complex pairs with Igaplha and Igbeta now forming the B cell receptor (BCR) that sends the success signal into the cell
• that is the end of Ig gene rearrangement and the completion of the first stage of B cell development
• This is also an amplification process: as B cells go through development they are dividing so every successful rearrangement is amplified, increasing the number of cells containing successful rearrangements

Question 3

What occurs at the pre-BCR checkpoint?

Answer 3

• The heavy chain/SLC complex is embedded in the cell membrane, surrounded by Ig alpha and Ig beta
• next to it are assembled a whole bunch of enzymes: Syk, Lyn, PLCgamma2, Btk, they are all important for sending a signal from the surface by a sequential cascade into the nucleus
  “They get activated and phosphorylate stuff, other stuff gets recruited and that phosphorylates other stuff.”
• this ultimately leads to transcription factors going into the nucleus and altering gene expression
• There are many factors involved in getting a signal to transmit from the Ig to the cell: if one of these does not work the signal cannot be transmitted
• In particular you need a heavy chain, Igalpha, Igbeta, BLNK (an adaptor protein), Btk (an enzyme that transmits signals)

Question 4

What are are mutations that result in Agammaglobulinaemia?

Answer 4

• X-linked agammaglobulinaemia (85% of cases/ incidence x 1/250,000)
  → almost always occurs in males
  → mutations in Btk responsible for ~85% of patients who show up with no B cell and no antibody: nothing to do with gene rearrangement»_space; signalling
• µ heavy chain (5% / v. rare)
• Surrogate light chain (1% / ex. rare)
• Igalpha/beta (2% / ex. rare)
• BLNK (2% / ex. rare)

Question 5

What are symptoms of humans with agammaglobulinaemia without B cells?

Answer 5

• No or very little serum immunoglobulin of any isotype
• No circulating B cells but normal T cells
• Recurrent infections primarily Strep. pneumonia; Haemophilus influenza
• Recurrent otitis (ear infection) in children and sinusitis in adults
• XLA mean age of diagnosis is 3 years

Question 6

What causes XLA?

Answer 6

• due to mutations in Bruton’s Tyrosine Kinase (BTK)
• Phospholipase Cgamma2 (PLCgamma2) activation requires recruitment to the plasma membrane via tyrosine phosphorylated B-cell linker protein (BLNK)
• PLCgamma2 is tyrosine phosphorylated by Btk, which is crucial for activation of PLCgamma2
• Mutations in BTK cause XLA with over 600 unique, inactivating mutations identified
• Mutations occur along the entire gene with no single mutation occuring more than 3%
• Mutation maintained in population by de novo events

Question 7

What mutation leads to a loss of all T, B and NK cells?

Answer 7

Adenosine Deaminase (ADA)
– rare
→ this gene assists in the production of nucleotides: lymphocytes are rapidly mutating and require lots of DNA and hence are very sensitive to losses in nucleotide production

Question 8

What are some symptoms associated with developmental combined immune deficiencies?

Answer 8

• no or very little serum immunoglobulin of any isotype
• may be circulating B cells but not T cells or NK cells
• Recurrent viral, fungal and bacterial infections
• Usually lethal in early childhood

Question 9

What are some current therapies for patients with agammaglobulinaemia without B cells?

Answer 9

Intravenous immunoglobulin - IVIg or Intragam

• purified IgG from pooled human sera using alcohol fractionation
• contains all IgG subclasses and traces of IgM and IgA
• Dose is 400-600 mg/Kg once per month in clinic, takes 2-4 hours infusion
• Idea is to maintain IgG at 5 g/L at trough - normal is 12 g/L with t half life of 21 days
• costs US$50,000 per year
• Following 300 patients over 5 years, only 20% remained infection free
• 80% had ≥ 1 major infectious complication (pneumonia, sinusitis, chronic lung disease)
• extremely demanding on patients
• can’t change the antibodies received by the patients in any way

Subcutaneous immunoglobulin - SCIg

• self-administered weekly
• Dose is 100-150 mg/Kg (40 - 60 ml of 16% IgG solution for a 70kg patient)
• Abdomen, thigh and forearms
• Delivered via a small pump
• In use for approx 10 years, no major adverse reactions

Question 10

What are some vaccine preventable diseases?

Answer 10

Standard

• diphtheria
• Tetanus
• Pertussis (whooping cough)
• Poliomyelitis (polio)
• Measles
• Mumps
• Rubella
• haemophilus influenzae type b infections
• hepatitis B
• Influenza
• pneumococcal infections

Based on Risk

• cholera
• hepatitis A
• meningococcal disease
• plague
• rabies
• bat lyssavirus
• yellow fever
• japanese encephalitis
• Q fever
• Tuberculosis
• Typhoid
• varicella-zoster (chickenpox)

Question 11

What is the basis of vaccines?

Answer 11

antibody production: because you are vaccinated with a form of a disease you make antibodies to that, thus protecting you from the infectious form of the disease

most vaccines induce production of serum IgG and this lasts a very very long time

Question 12

How long B-cell memory persist in humans after smallpox vaccination?

Answer 12

A group in the US tracked down people who had been vaccinated up to 60 years previously with the small pox vaccine and checked their antibody levels.
Even 60 years after the vaccination patients still had protective levels of antibody.
This is without re-exposure/boost as there is no small pox in the world.

Question 13

How does the immune response react (briefly) to a protein antigen? (in the context of a vaccine)

Answer 13

• initial exposure to antigen induces secretion of IgM followed shortly by IgG
• this is a change in isotype or class of immunoglobulin
• change from IgM to IgG (or IgA or IgE) is called Class Switch Recombination because it changes the class of the immunoglobulin from M to G or A or E.
• each class of Ig has discreet properties - all specialised in different things
• re-exposure induces rapid, robust response with prolonged, high affinity IgG
• the secondary or memory response is of:
  → faster kinetics
  → higher affinity
  → greater magnitude than the primary response
  → already IgG - don’t go through IgM

Question 14

What are immunological features of CD-40 deficient patients at diagnosis?

Answer 14

• very poor at making IgA - virtually none
• very low levels of IgG
• normal IgM
• normal T cell (70%) and B cell (20%) numbers
• i.e. able to make antibody but can’t make the downstream ones appropriately
• can’t make memory cells
• many have super-normal levels of IgM
• CD40L is on the X chromosome

Question 15

What is the clinical presentation of Hyper-IgM syndromes?

Answer 15

• Clinical symptoms develop during the first or second year of life
• most common are increased susceptibility to infection including recurrent upper and lower respiratory tract infections by bacteria
• lung infections may also occur, caused by viruses such as Cytomegalovirus and fungi such as Cryptococcus
• Gastrointestinal complaints, most commonly diarrhea and malabsorption, have also been reported in some patients
• patients often have enlarged tonsils, spleen and liver, and enlarged lymph nodes
• autoimmune disorders may also occur in patients with HIM syndrome, manifestations include chronic arthritis, low platelet counts (thrombocytopenia), haemolytic anemia, hypothyroidism, and kidney disease

Question 16

How do you get a B cell to differentiate into an antibody-secreting plasma cell?

Answer 16

• Involves substantial remodelling of the intracellular organelles
• requires a highly modifies gene expression programme and is tightly regulated
• dysregulated differentiation causes disease - autoimmunity or immune deficiency
• so mostly only undertaken under strict “guidance” from CD4 T cells

Question 16

How do B cells move in the body?

Answer 16

B cells are continuously patrolling your body - they move only in the regions designated for B cells (e.g. in the White pulp in the spleen) and will hang around a while before going elsewhere (e.g. lymph node)

Question 17

How are lymphocytes organised?

Answer 17

• into discrete regions within the secondary lymphoid organs
• the spleen contains lymphocyte rich regions called “white pulp” that form along the blood vessels within the erythrocyte rich “red pulp”
• lymph nodes contain few erythrocytes and no red pulp

Question 18

What is the distribution of lymphocytes within the white pulp in the spleen?

Answer 18

• B cells and T cells are separated from each other
• All the T cells stick together in the centre
• The B cells stick together in a ring around the T cells (these cells have never seen an antigen)
• macrophages surround the B cells
• outside of this are some B cells that respond to antigens immediately with no help and no guidance - very short lived

Question 19

What are the two outcomes of B and T cell interaction?

Answer 19

1. Plasma cells: differentiation into short-lived plasma cells of low affinity. First antibody produced.
2. Continued B cell proliferation in follicles forms a recognisable germinal center. T cell dependent.

Question 20

Describe the movements of dendritic cells and naive lymphocytes in the absence of antigen.

Answer 20

B cells and T cells bounce around in their respective areas
Every now and again the B cells and T cells will touch each other without inducing a response
DC’s float around in the T cell area

Question 21

Describe the early stages of B and T cell activation.

Answer 21

1. Antigen enters lymphoid organ, intact into B areas, processed to T
2. Antigen specific B and T cells are activated by Ag directly or after presentation by DC, which alters chemokine expression: T cells express CXCR5, B cells express CCR7
3. Activated B and T cells migrate towards each others areas, meeting at the boundary of T and B cell areas

Question 22

Transition to plasma cell is blocked by the lack of which transcription repressor?

Answer 22

Blimp1: a transcription repressor that shuts off the B cell expression programme and permits the plasma cell programme

Question 23

Continued B cell proliferation and the development of antibodies with higher affinity for the antigen is blocked when you lack which transcription repressor?

Answer 23

Bcl6: transcription repressor that promotes cell cycling and inhibits the response to DNA damage (SHM, CSR)

Question 24

What is the composition of a germinal centre?

Answer 24

- B cells (90%) - CD4+ helper T cells (5%) - Follicular dendritic cells (1%)

Question 26

What is going on within GCs?

Answer 26

1. Clonal expansion: T cell driven proliferation of Ag-specific B cells 2. Isotype switching: changing the constant region of the antibody heavy chain without changing the V region and thus the specificity. Changes effector function. 3. Somatic hypermutation: random introduction of point mutations into the V gene segments of the H and L chains to diversify binding to antigen 4. Affinity maturation: select those mutated B cells with improved affinity for antigen. Let the remainder die. Improves average affinity. 5. Memory formation: induce high affinity GC B cells to differentiate into either memory B cells or plasma cells that leave the GC and live for long periods

Question 27

What mutation/s lead/s to a loss of T and NK cells but not B cells?

Answer 27

gamma-c chain - cytokine receptor - rare JAK3 - signalling molecule downstream of cytokine receptor - v. rare

Question 28

What mutation/s lead/s to a loss of T cells but not B and NK cells (directly)?

Answer 28

IL7Ra (other part of cytokine receptor) – v. rare | This signalling sequence is what amplifies the lymphocytes as they go through the process of development

Question 29

What mutation/s lead/s to a loss of T and B cells but not NK cells?

Answer 29

RAG1/2 – rare Artemis – v. rare affect the actual rearrangement of genes themselves