Immune System Escape

Question 1

What are contagious cancers?

Answer 1

Masters of immune escape
EX - CTV1 in dogs,
- DFT1&2 in tasmanian devils: aggressive necrotic facial tumours transferred by bites.

Question 2

Basics of the innate and adaptive immune response

Answer 2

INNATE - phagocytes, inflammatory cytokines and cytokines with no immunological memory
ADAPTIVE - T Cells, B cells and antibodies with immunological memory

Question 3

How was it found that the immune system could recognise cancer cells?

Answer 3

Mouse immunised with irradiated tumour cells
An injection of viable cells from the same tumour produced antibodies and eliminated it
No response when cells from a different tumour were added, tumour growth.

Question 4

What parts of the immune system can kill tumours?

Answer 4

T cells, B cells and NK cells.

Question 5

What are T-cells?

Answer 5

cell-mediated adaptive immunity
kills viruses and bacteria
TCR surface receptors that recognise malignant cells through MHC on APCs.

• CD4+ = HELPER. TCR, CD3 + CD4 surface molecules. Secrete IFNy and interleukins.
• CD8+ = KILLER. TCR, CD3 + CD8 surface molecules. Secrete perforin, granzyme and IFNy.

Question 6

What are the two types of MHC?

Answer 6

1. CLASS 1 MHC – Found on ALL cells, presents endogenous antigens. Displays self-proteins, virus proteins and intracellular pathogens. Presents the antigen to CD8 cytotoxic T cells.
2. CLASS 2 MHC – Found on APCs, presents exogenous antigens. Used in phagocytosis and receptor mediated endocytosis. Presents to CD4 helper T cells.

Both MHCs need to be activated in an immune response. APCs express both types.

Question 7

Describe the peptide presentation pathway of a tumour specific antigen.

Answer 7

• Mutation is generated and translated to a protein
• Proteasome degrades proteins
• Peptides pumped into the ER by TAP (transport for antigen processing)
• ER chaperones and MHC class I bind specifically
• Peptide-MHC complex trafficked through Golgi to the PM to bind TCRs

Question 8

What is Co-stimulation and why is it required?

Answer 8

CO-STIMULATION is required for T-Cell cytotoxicity – T-cell activation need both the antigen presented on the MHC AND a co-stimulatory signal from an APC. This stops T cell killing at low levels of MHC.

Question 9

What are the 4 examples of how tumour antigens can arise, with specific examples for each.

Answer 9

High Specificity:

1. MUTATIONS - Proteins that are mutated are presented on MHCI (p53)
2. TUMOUR SPECIFIC EXPRESSION - peptides not usually expressed are expressed due to an epigenetic change and presented on MHCI (MAGE)

Low specificity:

3. TISSUE SPECIFIC EXPRESSION - peptides expressed in specific differentiated cells are presented and cause an immune response (MART1 melanomas)
4. OVEREXPRESSION - peptide is over expressed and presented more than usual in tumour cells (hTERT)

Question 10

3 cytokines examples.

Answer 10

• Interferon Gamma (IFNy) – the major inflammatory cytokine. Boosts MHC expression and stimulates CD4 and CD8 T-cells.
• Interleukin 2 (IL2) and 1 (IL1) – Stimulate clonal expansion of T-cells and allows APCs and CD4 cells to talk.
• Interleukin 12 (IL12) – Released by APCs and stimulated CD8 T-cell differentiation and IFNy production.

Question 11

What do NK cells need to kill?

Answer 11

Need both an activating signal and a loss of an inhibitory signal
Activating = stress protein produced by tumour
Inhibitory = MHC class I, sign of healthy self

Question 12

How are tumour specific antibodies produced and how are they involved in ADCC and CDC?

Answer 12

T-helper cells help B-cells produce tumour specific antibodies
ADCC - Antibodies specific for tumour antigens bind tumour cells so cells with Fc receptors can recognise the antibody and target the cell for destruction.
CDC - Antibodies specific for tumour antigens bind the tumour cells. This recruits C1q complement complex which releases membrane attack complex. Also recruits macrophages and NK cells.

Question 13

What are the different MHC genes?

Answer 13

Classical - HLA-A,B&C (polymorphic)
Non-Classical - HLA-G,E&F
Each HLA can specifically bind 100s of peptides

Question 14

What happens when the immune system finds it hard to distinguish between self and tumour antigens?

Answer 14

Conservative activation - tumour proliferation

Non-conservative activation - autoimmunity

Question 15

What is Burnett’s theory and an experiment to prove it?

Answer 15

The theory of immunosurveillance - cancer cells are recognised and eliminated by the immune system

The Shankaren experiment
RAG (-) mice has no antigen receptors and no lymphocytes. Tumours were innoculated in RAG(-) and WT mice. RAG (-) mice showed much more rapidly grower tumours and more aggressiveness due to the complete lack of immune system.
Similar trends were seen with IFNGR (-) and STAT1 (-)

Question 16

Why is perforin important in tumour regression?

Answer 16

Perforin is required for killing in CD8+ T-cells and NK cells.
Perforin knockout mice were more susceptible to p53 lymphomas than WT mice – shows the importance of cytotoxicity.

Question 17

What parts of the adaptive immune system were shows to be important in tumour regression and which weren’t?

Answer 17

A lack of perforin, CD8 T-cells and NK cells showed increased tumour progression.
A lack of NKT cells didn’t show this, not important in this process.

Question 18

How can immune cell infiltration be used as a prognostic marker?

Answer 18

COLD = no tumour cell infiltration
HOT = infiltration

Immunoscore - Tumour infiltrating lymphocytes are counted in colorectal cancers to see how many have inflitrated and create a prognosis score. More T-cells (and more in the centre of the tumour) showed a better survival rate.

Question 19

How can tumour specific antigens be identified and how can tumour infiltrating T-cells be used in immunotherapy?

Answer 19

Attach fluorophores to MHC tetramers to identify antigen specific T-cells. (e.g. MART1, gp100 in melanomas)
1 – Isolate the TILs from an isolated tumour.
2 – Make MHC tetramers with specific alleles.
3 – Screen the isolated cells and take the T-cells that are specific.
4 – Expand those specific T-cells and inject them back into the patient.

Question 20

Describe the new ways of identifying peptide antigens.

Method using functional analysis to create tumour specific T-cells.

Answer 20

• NGS allows new tumour antigens to be identified.
• Sequence tumour transcriptome
• Immunopeptidome sequenced by mass-spec (peptides MHC can express)
• Functional assays to test T-cell ability to respond to MHC-peptide complexes
• Analyse the tumour specific mutations. Predict epitopes that could be used on T-cells. Screen with MHC multiuser tech and create specific T-cells with specific epitope for TSA.

Question 21

Why are tumours such an immune problem?

Answer 21

Tumours are essentially ‘self’  sometimes their proteins are not immunogenic enough to produce an immune response. Tumours are also subject to evolutionary pressures and can become resistant to radiation and chemotherapy.

Question 22

Describe the Darwinian theory of cancer development.

Answer 22

Cancers evolve based on selective pressures to drive tumour natural selection.
The single progenitor stem cell becomes a mutated malignant cell which then produces many sub-clones, one will be able to survive a selective pressure (mutation) and repopulate the tumour.
Builds up a pattern of heterogeneity where tumours contain many different genomic subtypes within them.

Question 23

Describe the 3 steps of immunoediting process.

Answer 23

Elimination – Tumour cells are targeted by immune system, tumour cells destroyed.
CD4+ T-cells displaying tumour specific antigens.
Equilibrium – Tumour cells are controlled, but not fully eliminated by the immune system.
Tumour dormancy and cell editing. Some clones are still being picked off.
Escape – Tumour cells acquire new mutations that allow them to escape the immune system.
Checkpoint blockades, NK cell inhibitor mutations, poor immunogenicity of CD8+ cells.

Question 24

Describe an experiment that proved immunoediting.

Answer 24

MCA tumours grown in WT and RAG (-) mice.
Tumour cells grown in both mice were transplanted, RAG (-) tumours showed some rejection (unedited progressor or regressor), WT tumours did not (edited progressor).

Proves immune system edits tumours to make the less immunogenic.

Question 25

How are tumours edited? (5)

Answer 25

1. Total Loss - All MHC removed. Not a selective advantage because NK cells need the inhibitory signal to show that cells are functioning normally.
2. Selective Loss - Certain HLA genes that are able to present tumour specific antigens are lost.
3. Haplotype Loss - Loss of one allele of HLA genes. Becomes homozygous HLA.
4. Total Downregulation - Decreased expression of MHC class 1 and decreased tumour recognition.
5. Selective Downregulation - Decreased expression of HLA genes that can express tumour specific antigens.

Question 26

What are the two types of mutations that can affect MHC?

Answer 26

HARD - DNA mutations, irreversible (frameshift)

SOFT - Epigenetic changes, histone conformational changes, reversible

Question 27

Adaptive immunity maintains occult cancer in an equilibrium state, Koebel et al. Nature, 2007

Answer 27

• This paper showed that the adaptive immune system maintains occult (no signs or symptoms) cancer in the equilibrium state of immunoediting.
• Tumours are stable masses in equilibrium.
• More proliferation in progressive sarcomas, but stable masses still showed some proliferation.
• Any tumours in the equilibrium phase have an unedited phenotype.
• Adaptive IS knockouts, showed AIS most important for keeping tumour in equilibrium phase.

Question 28

Allele-Specific HLA loss and immune escape in lung cancer evolution, McGranahan et al. Cell, 2017

Answer 28

• HAPLOTYPE LOSS of either maternal or paternal allele. - Tumour cell can only present half of the possible peptides impact on presentation of TSAs.
• The allele lost can be pinpointed, provides phylogenetic tree and shows that LOH is not random.
• Class 1 alleles are lost by parallel evolution and HLA genes that bind TSA are more likely to be lost.
• Shows why non-small cell lung cancers have a fast onset once LOH occurs.

Question 29

An evolutionarily conserved function of Polycomb silences the MHC class 1 antigen presentation and enable immune evasion in cancer, Burr et al. Cancer Cell, 2019

Answer 29

• Transcriptional downregulation of HLA is more common that structural mutations.
• Polycomb recessive complex (PRC2) recruits histone markers to silence genes and down regulate.
• When PRC2 is knocked out, MHC class 1 can be restored.
• Inhibition of PRC2 restores T-cell killing function.
• PRC2 is responsible for MHC class 1 loss in transmissible tumours.