Topic 5 - Tumour Immunology

What are the 6 non-leukocytes involved in the TME?

mesenchymal, lymphatic endothelial cells, vascular endothelial cells, pericytes, fibroblasts, adipocyted

What are the 4 leukocytes involved in the TME?

TAMs, MDSCs, TANs, TDmDCs

What are the 3 lymphoid cells involved in the TME?

T, B and NK cells

What is the role of mesenchymal cells in the TME?

give rise to the TME (pericytes, fibroblasts, adipocytes, smooth muscle cells)

What is the role of lymphatic endothelial cells in the TME?

secrete VEGF, mechanically modulate the TME, alter host immune response

What is the role of vascular endothelial cells in the TME?

stimulated by angiogenic factors, result in leaky vessels

What is the role of pericytes in the TME?

increase hypoxia, poor prognosis, increased metastasis

What is the role of fibroblasts in the TME?

organ fibrosis, secrete tumour promoting factors

What is the role of adipocytes in the TME?

secrete adipokines & GF, provide FAs for fuel

What is the role of TAMs in the TME?

secrete VEGF + others, produce angiogenic factors, turn into M2 macrophages

What is the role of MDSCs in the TME?

can differentiate into TAMs, produce lrg amounts of IL-10, inhibit CD8+ T cells, polarise macrophages, induce Tregs

What is the role of TANs in the TME?

promote angiogenesis, increase ECM degradation, increase immune suppression, promote metastasis

What is the role of TDmDCs?

defective in TME, cannot adequately stimulate the immune system, some DCs suppress T cell

What T cells promote tumours?

Th2, Th17, Tregs

What T cells inhibit tumours?

CTL, Th1, ydT cells, NK T cells

What is the role of Th2 cells in the TME?

produce IL-4,5,13, sometimes is protective

What is the role of Th17 cells in the TME?

produce IL-17F,21,22, sometimes protective

What is the role of Tregs in the TME?

produce IL-12, TGFb, cell mediated contact

What is the role of CTLs in the TME?

can kill tumour cells

What is the role of Th1 T cells in the TME?

produce IL-2 & IFN, support CTL

What is the role of ydT cells in the TME?

cytotixic activity

What is the role of NK T cells in the TME?

potent producer of cytokines, support inflamm, promote anti-tumour benefits

What is the role of B cells in the TME?

Breg make IL-10, influence cells from outside of the TME, inhibit CTL, inhibit myeloid cells, may stop the tumour in some cancers

What is the role of NK in the TME?

infiltrate tumour stroma, anergic phenotype, induced by TGFb

How do CD8+ T cells and NK cells lead to the death of tumour cells? what cell starts the process, what cells are activated, what receptors are involved, what are the methods to kill

DCs recognise DAMPs via PRR, activate CD4+ & CD8+ T cells against tumour Ag --> CD8+ T cells recognise tumour Ag via MHCI & kill via granzyme/perforin, TRAIL, Fas-L --> NK cells activated by IFN-y (&IL-2), kill tumour cells bearing NKG2D ligands (stress associated molecule) using the same mechanisms as CD8+ T cells

What are 6 methods used by innate immune cells to kill tumour cells?

superoxide, NO, phagocytosis, cytokines, TNFa, IFN-y

What are 6 methods used by adaptive immune cells to kill tumour cells?

cytolysis, Ab, cytokines, TNFa, IFNy

What is immunosurveillance?

recognition and destruction of altered self

What is immunoediting?

cancer cells adapting phenotype to escape being killed by the immune system

What are the 2 mechanisms of immune evasion by cancer cells?

intrinsic (properties of tumour cells inhibit immune cell function) & extrinsic (products of tumour cells & TME inhibit immune cell function)

How do cancer cells intrinsically evade the immune system? (3)

against T cell killing - Lose tumour Ag and/or MHC class 1, lose cytokine receptors, express PD-L1 (binding PD-1 on T cells & killing them)

How do cancer cells extrinsically evade the immune system?

- attract cells that suppress immunity - myeloid derived suppressor cell, M2 macrophage, Treg

- tumour secreted factors

What cells do cancer cells attract to suppress immunity?

- T cells suppressed by Treg - release suppressor cytokines, IL-2 consumption (via IL-2R)

- DCs suppressed by Treg - CTLA-4 binds CD80 on DC --> decreases costimulation

- suppression by MDSCs - cytokines induce myeloid precursor --> MDSC, self-amplifying (S100A8), produces IL-10, Arg-1 and TGF-b

- suppression by M2 macrophages - Th2 prod IL-4,13, prod M2 macrophages, produce pro-tumour factors

What are 5 tumour secreted factors?

- TGFb - inhibits T cells, cell cycle arrest

- Galectin - inhibits T cell function

- Endothelin - vasoconstriction + inhibits leukocyte extravasation

- Arginase (Arg-1) - depletes arginine

- IDO & TDO - deplete tryptophan (starve T cells of crucial AA)

What are the 3 E's of cancer development? + evidence for 2 of them

Elimination --> equilibrium --> escape

- Equilibrium - transplanting kidney, got same carcinoma (as recipient's immune system was suppressed), same when artificially induce stable tumour in mice then deplete their immune system

- Escape - tumours who have lost Ag following immunoediting --> outgrowth & resistant to immune attac

What are the 5 pillars to cancer therapy?

surgery, radiotherapy, chemo, targeted therapy, immunotherapy

What are the 7 steps in the cancer immunity cycle?

1. Release of cancer cell Ag (cancer cell death)

2. Cancer Ag presentation (DC/APC)

3. Priming & activation (APCs & T cells)

4. Trafficking of T cells to tumours (CTLs)

5. Infiltration of T cells into tumours (CTLs, endothelial cells)

6. Recognition of cancer cells by T cells (CTLs, cancer cells)

7. Killing of cancer cells (immune & cancer cells

What is important to remember about the TME? (3)

- not just cancer cells, immune cells too (+ others)

- everyone's TME is different

- everyone's immune systems are different

What are the 2 forms of immunotherapy?

active / passive

What are 4 types of mAb used to treat cancers + their MOA?

- inhibiting angiogenesis - against VEGF, against VEGF receptors, against downstream pathways

- drug-Ab conjugates - bind to cancer, endocytose, toxin released

- radioimmunotherapy - use Ab to guide radionuclide to specific cells

- bi-specific Ab - bring APC, T cell & tumour cell all together, ADDC & phagocytosis from APC + lysis from T cell

What is checkpoint blockade?

inhibit the ability of cells to become exhausted, inhibiting the brakes, allow cells to stay activated for longer

What do anti-CTLA4 mAb do? + example

priming & activation of T cells (stop T cells from saying they are exhausted), eg ipilmumab

What do anti-PD-1/PD-L1 mAb do? + example

killing of cancer cells (stop cancer cells from telling them they are exhausted), eg nivolumab

Why is using one checkpoint blockade not enough?

the tumor compensates through other mechanisms to generate resistances and reduce the efficacy

What are 3 examples of combination therapy?

- as big activation of T cells/enhances immune activity

- targets two arms of cycle

- depletes TME Tregs (eg anti-CTLA4 can target Tregs at TME)

What are 6 disadvantages to combination therapy?

- increase adverse effects

- need pre-existing immune response (eg T cells)

- not all cancers express PD-L1

- primary impact of anti-CTLA4 drug is in 2ndary lymphoid organs (issue if they then get blocked at tumour site)

- issue w/ gut microbiome (det how patients respond to therapy)

- cancer may then induce other checkpoint inhibitory molecules

What are the 5 steps to adoptive T cell immunotherapy?

- take tumour

- fragment cells

- grow & activate T cells

- expand the population

- reinfuse

What are CAR-T cells made of?

T cells have been designed with Ab domain (targets, from BCR) and signalling domain (increase ability to promote T cell activity, from TCR) and also can carry pay loads (eg promote IL-12 production)

What are 5 general types of CAR-T cells?

- 3 generations

- TRUCKS (have payloads)

- suicide CARs - want to limit T cells, use exogenous addition to stop T cells

- dual CARs, bispecific CARs

- TCR-mimic CARs

How are CAR-T cells made? (4)

Transfect packaging line --> creates viruses --> add viruses + activated human T cells --> create CAR-T cells

What are the disadvantages of CAR-T cells?

poor trafficking, poor expansion, limited persistence (levels of CAR-T cells correlate w/ patient survival), toxicity-related adverse effects (insertional oncogenesis, neurological toxicity, "on-target, off-tumour" toxicity, anaphylaxis/allergy, cytokine release syndrome)

What remains an issue w/ CAR-T cells?

these cells also become exhausted due to checkpoint inhibition. Thus, use checkpoint blockage in combination

What are the 2 approaches for overcoming exhaustion of CAR-T cells?

extrinsic (separate checkpoint inhibitors) or intrinsic

What are the 3 methods for intrinsic checkpoint inhibition of CAR-T cells?

- dominant negative receptor: Remove ability for downstream signalling (internal part of PD-1 receptor)

- ScFv secretion - specifically secrete PD-1 blocking scFv to inhibit PD-1

- gene editing - use CRIPSR/Cas9 system to completely delete PD-1

What are 2 key points for future immunotherapy?

- combination therapy + tailored for each patient & their TME

- personalised immunotherapy = genetic, molecular & immune (& microbiota) profiling

What medical discoveries have been made in wild and comparative immunology?

- camel Ab and nano Ab

- new types of T cells in marsupials, wild mice imm show enviro det number & level of activation of CD8+ T cells

- conventional imm & that IgM/G associated with external Ag contact (difference between lab mice & wild hyeenas)

What are 4 issues w/ wild imm?

lab conditions not ideal, hard to perform controlled experiments, time course experiments are hard, lack of reagents

What is DFT an example of?

1 of 2 types of contagious cancers

What does DFT1 not express?

DFT1 cells do not express MHC-1 due to genetic & epigenetic silencing of MHC-1 (hemizygous deletion of B2M in DFT1 ) --> it is an intrinsic immune evasion technique

What is a major target for Ab against DFT1?

MHC-1

Inducing MHC class I expression makes the tumour cells more "visible" to the immune system and can be used in immunotherapy

How can you upregulate MHCI in DFT1?

IFNy

What are the immune responses observed in devils vaccinated w/ DFT?

increases in MHC class II and CD3 in the tumour microenvironment of vaccinated animals. Immune cells that are likely to be found here are APCs and T cells (as MHCII are expressed APC and CD3 on TCR complex)

Is PD-L1 always expressed by DFT cells?

D-L1 isn't always expressed by DFT cells, but if expression is induced, then it could be used as an immune evasion mechanism

What is the FAST system?

the FAST protein system is important as it can be used to generate new reagents to study the immune system in a range of species including the Tasmanian Devil.

What can inject & express transgenes in DFT cells?

human adeno virus

What will the DFT vax do & how will it do it?

- he oral bait vaccine for DFT is postulated to be both preventative (stops tumour development) and be effective as immunotherapy (to treat existing DFT).

- the adenovirus used as part of the oral bait vaccine will be engineered to express IFNgamma as a way to overcome the immune evasion strategy used by DFT (e.g that these cells don't express MHC class I) + encode devil facial tumour Ag + activate & educate the immune system

What 4 things are needed for a good vax?

immunogenic delivery system, targets for immune cells, assessment of efficacy, funding

What are the can the DFT vax do, what are the targets, and the program's objectives?

- immunotherapy can activate & educate the immune system

- targets for immune cells --> mutation derived neoAg (aka non-synonymous variants)

- objectives - safety trials --> immunotherapy trials --> prevention trials

What are the 2 places that haematological malignancies arise?

From cells that are highly proliferative

- either during development (e.g. those from progenitors - AML and ALL)

- when activated in the periphery (e.g. those from mature cells - CLL, lymphoma, myeloma).

What are the 3 lineages implicated in haematological malignancies?

May be myeloid lineage, B lineage or T lineage cells.

What are lymphomas classified as?

solid tumours (not really leukaemia)

What is the common mutation in haematological malignancies?

TF

What determines treatment & prognosis of haematological malignancies?

differential diagnosis

What are 4 symptoms to haematological malignancies?

fatigue, infection, spleen & lymph node enlargement

What are 4 ways haematological malignancies can be identified?

blood count, cell morphology (blood smear, bone marrow aspirate), flow cytometry, genetics (karyotyping/sequencing)

What are the 2 ways haematological malignancies are classified?

lymphoid vs myeloid, acute vs chronic

What is of note w/ AML & CLL?

malignancies of myeloid and lymphoid progenitors, respectively, there has been a differentiation block at the blast stage of development, blood smears in these cancers look morphologically similar, being large (both the cell and nucleus)

Where do other blood cancers arise from?

other blood cancers arise from mature cells - B cell lineage cancers such as CLL and myeloma (PC malignancy), while lymphoma can arise from both B cells and T cells.

What are blood counts/smears good for & not good for?

good for leukemias for AML, but some cancers are localised to BM or lymphoid organs (blood count not useful here)

What is the abundance profile of WBC?

neutrophils, lymphocytes, monocytes, eosinophils, and basophils ("Never Let Monkeys Eat Bananas (NLMEB))

What do TF control? (5)

differentiation, function, survival, proliferation, identity

How is haematopoiesis controlled?

○ Control w/ intrinsic (TF) & extrinsic (cytokine) inputes

What are the master TF for the key lineages?

PU.1, CEBPA (myeloid lineage), NOTCH-1 (T cell lineage), PAX5, E2A, EBF1 (B cell lineage)

What is ALL?

acute lymphoblastic leukemia

- most common childhood cancer

- 2-5yrs (a lot of adaptive immune prolif)

- B or T cell lineage

What are the 3 therapies for ALL?

glucocorticoid, chemo, asparaginase (dramatic improvement in last 50yrs, better dosage & scheduling)

toxic side effects, need >2yrs

cure rate 90%, relapse often fatal

What is the blood like of patients w/ ALL?

milky

What are 2 similarities of B-ALL and T-ALL?

- differentiation block @ lymphoblast stage

- pre-BCR/TCR after V(D)J recombination

What is the process of B-ALL? eg where is it, what TF are involved

- bone marrow, LOF, eg PAX5, EBF1

- 30% of B-ALL cases = PAX5

- monoallelic (point mutation)

- reduced PAX5 protein levels &/or activity

- germline PAX5 mutation = susceptability

- total loss = no B cells at all

What is the process of T-ALL? eg where is it, what TF are involved

- thymus, GOF, eg LMO2, NOTCH1

- NOTCH1 req for thymocyte prolif & dev

- activates prolif genes including Pre-T cell receptor alpha (PTCRA) & MYC

What is the process by which errors in normal lymphoid dev leads to cancer?

- defective VDJ recomb

- B lineage - IgH & IgL loci

- T lineage - TCRy/d loci??

- RAG1/2 nuclease --> NHEJ

- oncogenic translocation can result from unresolved V(D)J recombination (by products of mistakes in this process)

What are 4 lymphomas due to issues w/ normal lymphoid dev?

- BCL6 (transcription) = Diffuse Large B Cell Lymphoma (DLBCL)

- MYC (proliferation) = Burkitt Lymphoma

- CYCLIN D1 (proliferation) = Mantle Cell Lymphoma

- BCL2 (survival) = Follicular Lymphoma

What have mouse models shown us in regards to V(D)J recombination issues?

- unresolved Rag-mediated DNA breaks normally lead to p53-dependent apoptosis of lymphoid progenitors. If p53 is mutated these cells can survive persistent DNA damage signaling and are prone to sustaining oncogenic translocations

What is CLL & what are the normal mutations that cause it?

chronic lymphocytic leukemia

all converge on quite similar processes - Notch signalling, inflamm pathway, BCR signalling & differentiation, Wnt signalling, DNA damage & cell cycle, chromatin mods, RNA & ribosomal processing

How many lymphomas are Hodgkin's?

10%

What portion of NHL are DLBCL?

35%

What is DLBCL?

diffuse large B-cell lymphoma

a diverse collection of lymphomas, driven by a diverse range of mutations & transcriptional changes

some have GOF, some have LOF

How else can DLBCL be differentiated?

activated & GC, look the same using flow cytometry, have different transcriptomes

What is ABC?

active BCR signalling, sensitive to BTK inhibition, self Ag drive (self glycoprotein, self Ag from dying cells, self idiotope)

What is GCB?

tonic BCR signalling, 45% have IgH-BCL2 translocation, no Ag that activates

What is multiple myeloma?

- clonal disorder of PCs

- each PC produces a mAb, paraproetin / M protein

- MM --> paraprotein w/ additional myeloma defining criteria (all start w/ MGUS)

- 2 types of founder lesions - translocation (eg w/ IgH) or hyperdiploid