Immunology Week 10

What are the main types of lymphocytes?

B cells; CD4+ helper T cells; CD8+ cytotoxic T cells; Natural killer (NK) cells

What is the main function of CD8+ T cells?

Recognize antigens on MHC I and kill cancer cells via perforin and granzymes, inducing apoptosis

What molecules do cytotoxic T cells use to kill target cells?

Perforin and granzymes

What is the role of MHC class I in cancer immunity?

Presents tumour antigens to CD8+ T cells for recognition

Why are T-cell therapies less effective in solid tumours?

Poor trafficking, lack of ideal antigens, and immunosuppressive tumour microenvironment

What are the steps in CAR-T cell therapy?

Extract T cells → genetically modify with CAR → expand → reinfuse into patient

Why is CAR-T therapy effective in blood cancers?

Cancer cells are more accessible and lack complex physical/immunosuppressive barriers

Why is CAR-T therapy less effective in solid tumours?

TME barriers, antigen heterogeneity, poor infiltration, immunosuppression

What is the tumour microenvironment (TME)?

A complex system of cancer cells, immune cells, ECM, vasculature, and stromal cells

What is a key feature of the TME?

It is heterogeneous and actively suppresses immune responses

How do tumours use immune cells in the TME?

Recruit and convert them into tumour-supporting “bodyguards”

What causes hypoxia in tumours?

Rapid growth outpaces blood supply

What is the effect of hypoxia in tumours?

Creates a dead core and reduces T-cell survival

How does ECM affect T-cell function?

Acts as a physical barrier preventing infiltration

What is abnormal about tumour vasculature?

Leaky, disorganized, impairs immune cell trafficking

What is the effect of low chemokines in TME?

Reduced immune signalling and T-cell activation

How do CD8+ T cells recognise tumour cells?

TCR binds tumour antigen presented on MHC I

What is T-cell infiltration?

Movement of T cells into tumour tissue

Why is T-cell infiltration difficult in solid tumours?

Physical barriers, hypoxia, abnormal vasculature, low chemokines

What is T-cell exhaustion?

Dysfunctional state due to chronic stimulation with reduced cytokine production and activity

What cytokines decrease in exhausted T cells?

IL-2, IFN-γ, TNF-α

What is T-cell anergy?

State of unresponsiveness where T cells cannot activate even if antigen is recognised

What is metabolic competition in TME?

Tumour consumes nutrients and oxygen, starving T cells

What is tumour antigen heterogeneity?

Tumour cells express different antigens, making targeting difficult

What is antigen escape?

Tumour cells lose or alter antigens to avoid immune recognition

What cytokines do tumours release to suppress immunity?

IL-10 and TGF-β

What is CSF-1 role in tumours?

Recruits tumour-associated macrophages (TAMs)

What is the role of STAT3 in tumours?

Promotes immunosuppression, angiogenesis, and tumour growth

How does hypoxia affect signalling pathways?

Increases VEGF and activates STAT3

What is VEGF?

Vascular endothelial growth factor that promotes abnormal blood vessel formation

How do tumours reduce antigen presentation?

Downregulate MHC expression

What are immune checkpoints?

Regulatory proteins that prevent autoimmunity by inhibiting T-cell activation

What are key immune checkpoints in cancer?

PD-1, PD-L1, CTLA-4

How do tumours use immune checkpoints?

Upregulate checkpoint ligands to suppress T cells

What are immune checkpoint inhibitors (ICIs)?

Drugs that block checkpoint interactions and restore T-cell activity

What are the three categories of tumour immune evasion?

Antigen loss, T-cell inhibition, immunosuppressive environment

What is the role of Tregs in tumours?

Suppress T-cell responses

What are MDSCs?

Myeloid-derived suppressor cells that inhibit anti-tumour immunity

What is tumour immune evasion?

Process by which tumours avoid immune detection and destruction

What are general inflammation pathways in cancer therapy?

Strategies that activate immune responses via cytokines or antibodies

What is ADCC?

Antibody-dependent cell-mediated cytotoxicity

What is CCL21 role in therapy?

Recruits lymphocytes and dendritic cells to tumour

What are nanoparticle therapies?

Delivery systems that improve stability and targeting of drugs/cytokines

What is TIL therapy?

Extraction, expansion, and reinfusion of tumour-infiltrating lymphocytes

How does CAR-T therapy cause toxicity?

Can attack healthy tissues (off-tumour toxicity)

What is antigen heterogeneity problem in CAR-T?

Different tumour cells express different targets

What are next-generation CAR-T improvements?

Better specificity, persistence, reduced toxicity

What is combination immunotherapy?

Using multiple treatments (e.g. CAR-T + ICIs) to improve outcomes

What is TME modulation?

Altering tumour environment to enhance immune function

What is CCR5 role in CAR-T therapy?

Improves T-cell migration into tumours

What is IL-12 role in CAR-T therapy?

Enhances T-cell activity and counteracts immunosuppression

Why combine CCR5 and IL-12?

Improves both infiltration and function of T cells

What are main challenges in solid tumour therapy?

Poor infiltration, antigen variability, immunosuppression, toxicity

What is the best strategy for treating solid tumours?

Combination therapies targeting multiple mechanisms

What is the key takeaway about T cells in solid tumours?

They are effective but suppressed by the tumour environment

What is the main focus of Lecture 2?

Imaging T-cell function and tracking T-cell behaviour in tumours

Why is imaging T cells important in cancer therapy?

To track location, infiltration, and persistence and evaluate therapy effectiveness

What are the key challenges in imaging T cells in solid tumours?

Dense structure, heterogeneity, and difficulty visualising infiltration

What are the three main challenges for T cells in the TME?

Physical barriers, T-cell dysfunction/exhaustion, antigen heterogeneity

What is meant by T-cell infiltration?

Movement of T cells into tumour tissue

Why is T-cell infiltration difficult in solid tumours?

Dense ECM, abnormal vasculature, and immunosuppressive environment

What is T-cell exhaustion?

Dysfunctional state with reduced responsiveness due to chronic stimulation

What is T-cell anergy?

Severe dysfunction where T cells become unresponsive ("inactive state")

What is tumour antigen heterogeneity?

Variation in antigen expression across tumour cells

Why does antigen heterogeneity limit therapy?

Makes it difficult to target all tumour cells with one strategy

What is the purpose of cell labelling in imaging?

To track cells in vitro and in vivo using fluorescence

What is fluorescence in cell imaging?

Emission of light after absorption of energy by π-conjugated systems

What are non-covalent dyes?

Dyes that bind weakly (e.g. membrane insertion) and can move or be lost over time

What are membrane dyes used for?

Insert into lipid bilayer to label and track cells

What are Vybrant dyes?

Long-chain carbocyanine dyes that insert into membranes for cell tracking

What are key features of Vybrant dyes?

Non-covalent, low cytotoxicity, transferable during cell division

How long can Vybrant dyes track cells?

Approximately 2–3 days

What is a limitation of non-covalent dyes?

Signal loss over time due to diffusion and cell division

What is Hoechst 33342 used for?

Nuclear staining in live cells

How does Hoechst dye work?

Binds to AT-rich regions in DNA minor groove and fluoresces

What is a limitation of Hoechst dye?

Can cause DNA damage and fluorescence decreases over time

What is the difference between Hoechst and DAPI?

Hoechst is cell-permeable (live cells), DAPI requires fixed cells

What are BODIPY dyes used for?

Organelle imaging and probes (e.g. Golgi, lysosomes)

What are advantages of BODIPY dyes?

High fluorescence, tunable properties, photostability

What is a limitation of BODIPY dyes?

Poor long-term retention and not transferred to daughter cells

What is covalent labelling?

Stable attachment of dyes to cellular proteins via chemical bonds

Why use covalent labelling?

Long-term retention and tracking across cell generations

What is maleimide labelling?

Covalent binding to thiol groups (cysteine residues)

What is NHS ester labelling?

Covalent binding to primary amines (lysine residues)

What is a disadvantage of covalent labelling?

Potential cytotoxicity and non-specific binding

What are CFSE and CFDA-SE dyes used for?

Tracking cell proliferation

How does CFSE track cell division?

Fluorescence halves with each cell division

What is CFDA-SE?

A non-fluorescent precursor activated inside cells

What is metabolic glycan labelling?

Labelling cells via incorporation of modified sugars into glycoproteins

What molecule is used in metabolic glycan labelling?

Ac4ManNAz (azide-modified sugar)

What reaction is used in glycan labelling?

Click chemistry (SPAAC reaction)

What is an advantage of metabolic glycan labelling?

More specific and less cytotoxic than protein labelling

What is a limitation of glycan labelling?

Signal decreases over 72–96 hours

What is flow cytometry?

Technique to measure physical and fluorescent properties of cells in flow

What does forward scatter (FSC) measure?

Cell size

What does side scatter (SSC) measure?

Cell complexity/granularity

What does flow cytometry measure in T-cell studies?

Fluorescence intensity, cell size, proliferation

How is proliferation tracked using flow cytometry?

Fluorescence intensity halves with each generation

What is confocal microscopy used for?

3D imaging of cells using optical sectioning (z-stacks)

What is an organoid model?

3D tumour model used to study T-cell infiltration and behaviour