TIL Therapy and Neoantigens: How Tumor-Infiltrating Lymphocytes Recognize Mutations

By Lociven · NeoantigenLab · July 2026

Figure 1. TIL manufacturing timeline (left) and neoantigen-reactive TIL frequency by tumor type (right).

TIL Therapy Series · Part 1 of 2

When Iovance's lifileucel received FDA approval in 2024 for metastatic melanoma, it validated TIL therapy as a viable commercial modality. But understanding why TIL therapy works — and why it sometimes doesn't — requires understanding the relationship between tumor-infiltrating lymphocytes and neoantigens.

This post explains how neoantigens shape the TIL compartment, what neoantigen-reactive TILs look like functionally, and what this means for TIL expansion protocols.

What TILs are and where they come from

Tumor-infiltrating lymphocytes are T cells (predominantly CD8+, but also CD4+) found within the tumor microenvironment. They are not randomly distributed — they are shaped by the antigens present in the tumor, including neoantigens.

When a somatic mutation generates a neoantigen that is presented on MHC, it can prime a T cell response in the tumor-draining lymph node. Those primed T cells traffic to the tumor, where they encounter their cognate antigen and attempt to kill tumor cells. This is the basis of natural anti-tumor immunity — and it is the T cells generated by this process that are collected, expanded, and re-infused in TIL therapy.

Neoantigen-reactive TILs: evidence and frequency

The evidence that TILs recognize neoantigens is now robust:

  • Rosenberg lab studies (NCI) identified neoantigen-reactive T cells in TIL products from melanoma, colorectal, and breast cancer patients
  • In melanoma, neoantigen-reactive T cells make up 1–5% of bulk TIL in most patients
  • In colorectal cancer with MSI-H (high neoantigen burden), neoantigen-reactive TILs can make up 10–20% of the infiltrate
  • The frequency of neoantigen-reactive TILs correlates with TMB and clonal neoantigen burden

The low absolute frequency (1–5%) is why standard bulk TIL expansion can dilute neoantigen-reactive cells — you expand everything, including bystander T cells, regulatory T cells, and exhausted cells that respond to viral antigens.

Identifying neoantigen-reactive TILs

Several approaches are used to identify and select neoantigen-reactive cells from a TIL product:

Peptide pool stimulation + cytokine capture: TILs are stimulated with pools of predicted neoantigen peptides. Cells that respond (IFN-γ secretion) are captured by anti-IFN-γ antibody and sorted. This is the most direct approach but requires peptide synthesis for each patient.

pMHC tetramer staining: For confirmed HLA-peptide combinations, tetramers allow direct staining and sorting of antigen-specific T cells. Highly specific but requires one tetramer per neoantigen-HLA pair — expensive for large candidate lists.

Activation marker enrichment (TETRAMER-free): After peptide stimulation, cells are stained for activation markers (4-1BB / CD137, OX40). 4-1BB upregulation after antigen stimulation is a reliable proxy for TCR engagement. This is more scalable than tetramer staining.

Single-cell TCR sequencing: scTCR-seq of TIL products can identify clonally expanded TCRs. Clonal expansions are enriched for antigen-specific cells. Coupling scTCR-seq with predicted neoantigen-HLA pairs allows retroactive identification of neoantigen-reactive clones.

Exhaustion: the TIL problem

TILs in solid tumors are chronically exposed to antigen in an immunosuppressive microenvironment. This chronic stimulation drives T cell exhaustion — a program of progressive dysfunction characterized by:

  • Upregulation of inhibitory receptors: PD-1, LAG-3, TIM-3, TIGIT
  • Loss of effector cytokine production (IL-2 first, then TNF-α, then IFN-γ)
  • Reduced proliferative capacity
  • Expression of transcription factors TOX and NR4A1 that lock in the exhausted state

In TIL therapy, the ex vivo expansion step (2 weeks in IL-2) partially reverses exhaustion by removing the tumor antigen stimulus. The re-infused cells are less exhausted than the original TIL — which is partly why ex vivo expansion improves function. But the exhaustion-related epigenetic marks persist, and TILs re-encountering tumor antigen in vivo tend to re-exhaust.

This is why lymphodepletion conditioning before TIL infusion matters: it creates homeostatic space and reduces competition from endogenous T cells, giving the infused TILs a survival advantage before re-exhaustion sets in.

Implications for neoantigen-guided TIL therapy

The current clinical approach (bulk TIL expansion as in lifileucel) is not neoantigen-guided — all T cells are expanded together and re-infused. This works in melanoma because melanoma TIL products tend to be rich in neoantigen-reactive cells to begin with (high TMB, highly immunogenic).

The next generation of TIL products aims to enrich for neoantigen-reactive cells:

  • Selected TIL: Pre-screen for neoantigen reactivity, expand only reactive cells
  • TCR-engineered T cells: Identify TCRs from neoantigen-reactive TILs, engineer peripheral T cells to express those TCRs
  • TIL + vaccine: Expand TIL, then boost with neoantigen vaccine to re-prime neoantigen-specific cells in vivo

These approaches are in early clinical development. The bottleneck is identifying which TCRs are neoantigen-reactive at scale — a problem that single-cell multi-omics is beginning to solve.

The final post in this series brings everything together: a complete walkthrough of the WES-to-neoantigen workflow, from raw sequencing data to a prioritized candidate list ready for experimental validation.

Tags: TIL therapy, tumor-infiltrating lymphocytes, neoantigen, lifileucel, T cell exhaustion, TCR, neoantigen-reactive TIL, cancer immunotherapy

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Tumor size criteria, vein-to-vein timeline (including how Iovance's fixed 22-day Gen2 process differs from standard manufacture), and neoantigen-reactive TIL enrichment steps — fact-checked against current clinical protocols.

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