BN - Tumour neoantigens as personalised T cell vaccine targets

Question 1

Q1: Where and how does T cell priming occur in response to tumour neoantigens? (4)

Answer 1

• T cell priming occurs in the tumour-draining lymph nodes (TDLN).
• APCs present antigens to naïve T cells, initiating activation.
• Activated CD4+ and CD8+ T cells proliferate and differentiate.
• These cells traffic to the tumour and infiltrate as tumour-infiltrating lymphocytes (TILs).

Question 2

Q2: How do cells present proteins to T cells using HLA molecules? (5)

Answer 2

• APCs ingest tumour cells, degrade proteins into peptides, and load them onto HLA.
• HLA-I: presents intracellular peptides (e.g., viral/tumour antigens) to CD8+ T cells; all nucleated cells.
• HLA-II: presents extracellular peptides (e.g., bacterial antigens) to CD4+ T cells; primarily APCs.
• HLA genes are polygenic (multiple genes) and polymorphic (many alleles), creating diversity.
• A person typically expresses 6 HLA-I and 8 HLA-II variants.

Question 3

Q3: What are the structural differences between HLA-I and HLA-II and how do they affect peptide binding? (5)

Answer 3

• HLA-I: Closed groove, binds 8–11 aa peptides; peptide must fit entirely.
• HLA-II: Open groove, binds 12–18 aa peptides; peptide can extend outside the cleft.
• HLA-I presents to CD8+ T cells, HLA-II to CD4+ T cells.
• APCs express both HLA-I and II to activate both T cell types.

Question 4

Q4: How do HLA-I and HLA-II cooperate in anti-tumour responses? (4)

Answer 4

• CD4+ T cell help is crucial for CD8+ T cell priming and function.
• Some tumours lack MHC-II but still rely on CD4 assistance for clearance.
• CD4+ help enhances CD8+ clone expansion and killing efficiency.
• Best immunotherapy outcomes involve targeting both HLA-I and HLA-II neoantigens.

Question 5

Q5: How are computational tools used to predict neoantigen-HLA binding? (5)

Answer 5

• Algorithms (e.g. NetMHC) predict peptide binding to HLA based on sequence motifs.
• Inputs include mutation data, gene expression, and HLA typing.
• Protein sequences are chopped into overlapping 8–11 (I) and 12–18 (II) aa peptides.
• Each peptide is evaluated for binding affinity to specific HLA allotypes.
• Tools like pVACseq combine binding predictions with expression data for prioritisation.

Question 6

Q6: Why is it difficult to predict functional neoantigens? (6)

Answer 6

• Easy to predict if a gene is mutated or expressed.
• Hard to predict if mutant protein is translated.
• Even harder to predict if it is processed into peptides by HLA machinery.
• Peptide may be too similar to self to trigger immune response.
• Central tolerance removes T cells that react to self-peptides.
• Only \~1–5% of predicted neoantigens trigger T cell responses.

Question 7

Q7: How does a tetramer assay measure neoantigen-specific T cells? (3)

Answer 7

• Fluorescent peptide-HLA tetramers bind specific TCRs on T cells.
• Analyzed via flow cytometry to determine % of T cells that bind the peptide.
• Doesn’t require APCs, but sensitivity is low due to scarce T cell populations in blood.

Question 8

Q8: How does an Elispot assay assess T cell activation by neoantigens? (5)

Answer 8

• Expose HLA-matched T cells to wild-type vs mutant peptides.
• Measures release of IFN-γ, indicating T cell activation.
• Uses restricted or long peptides.
• Highly sensitive and quantitative.
• Outcome: # of IFN-γ-secreting T cells per million input cells.

Question 9

Q9: What activation markers are used to detect T cell responses to neoantigens? (3)

Answer 9

• Markers: 4-1BB and Ox40 on CD8+/CD4+ T cells.
• T cells are exposed to predicted neoantigen peptides.
• Flow cytometry quantifies % of activated T cells expressing these markers.

Question 10

Q10: What are tandem minigenes and how are they used? (4)

Answer 10

• Minigenes: Synthetic DNA encoding a mutant peptide plus regulatory regions.
• Transfected into autologous APCs for natural antigen processing.
• Allow presentation of peptides via HLA-I/II, mimicking physiological processing.
• Problem: Difficult to determine which peptide triggered the response.

Question 11

Q11: What are the three neoantigen vaccine strategies and how do they work? (3)

Answer 11

DC-based vaccines:

• Autologous dendritic cells loaded with neoantigens ex vivo, injected back to activate T cells.

SLP vaccines:

• Synthetic long peptides injected, taken up and processed by APCs, presented to CD8+ cells.

RNA vaccines:

• Injected RNA encodes neoantigen peptides; translated in vivo and presented on MHC by APCs.

Question 12

Q12: What are the limitations of neoantigen vaccine approaches? (6)

Answer 12

• Tumour heterogeneity – mutations vary across tumour regions.
• Tumour immunosuppressive microenvironment prevents immune activation.
• Immunoediting – selective pressure removes immunogenic cells.
• Most neoantigens are from passenger mutations, not conserved drivers.
• CAR-T cells often fail in solid tumours due to poor infiltration.
• Often requires checkpoint inhibitors to unleash full T cell response.

Question 13

Q13: What are promising developments in the field of neoantigen immunotherapy? (5)

Answer 13

• Improved neoantigen prediction combining mass spectrometry and sequencing.
• Combination of neoantigen vaccines + checkpoint inhibitors shows promise.
• Potential to clone neoantigen-specific TCRs for use in engineered T cell therapies.
• Clinical trials ongoing to evaluate best vaccine formulation.
• Neoantigen vaccines represent a personalised, precise approach to cancer treatment.