Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients

Abstract

Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4⁺T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress.

Methods: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT.

Results: We discovered that neoantigen-specific TIL clones were predominantly CD4⁺ T cells and were present in both therapeutic responders and non-responders. CD4⁺ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4⁺ TIL.

Conclusions: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4⁺ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4⁺ TIL in future ACT protocols as a strategy to improve antitumor immunity.

Keywords: CD4-positive T-lymphocytes; antigens; immunotherapy, adoptive; lymphocytes, tumor-infiltrating; melanoma.

Figure 1

CD4⁺ TIL exhibit effector response on neoantigen peptide recognition. (A) Treatment course of ACT for patient 1, accompanied by CT images of target lesions (red circles) before and after therapy. D=day (relative to TIL infusion), Sx=surgery, TL=target lesion. (B) Measurement of the longest diameter of each target lesion (upper) and sum of target lesions (lower) during patient follow-up window. Red dotted line indicates a −30% change from the baseline sum. (C) All neoantigen peptides (n=65) were prioritized by additive score (AS). Blue arrow indicates peptide 60 (P60). See the Methods section for detailed explanation. ES=expression score, MHC-II=mhc2_ score, MHC-I=mhc1_score, (D) Bulk TILs were cocultured with DC loaded with the neoantigen peptide pool (PP) or no peptide (NP), then stained for cell sorting by OX40 and 4-1BB induction. (E) Sort coculture supernatants were assayed for TIL production of granzyme B (GZMB), IFNg, and TNFa via the Ella system. (F) TILs were validated for individual neoantigen peptide recognition by flow cytometric evaluation of OX40 and 4-1BB expression (upper) and IFNg release (lower) in coculture supernatants. (G) Enriched CD4⁺ TILs were assayed for effector molecule secretion in response to stimulation with P60 or no stimulation (TIL). (H) Mutant (P60^MUT=P60) and wildtype (P60^WT) versions of P60 were loaded onto APCs and used to stimulate enriched CD4⁺ TIL. IFNg release was quantified in coculture supernatants. (I) Following coculture of enriched TIL with P60 or NP, CD4⁺ TIL were stained for flow cytometric analysis of the indicated cell surface and intracellular molecules. ACT, adoptive cell therapy; APCs, antigen presenting cells; DC, dendritic cell; TIL, tumor-infiltrating lymphocyte.

Figure 2

CD4⁺ TILs demonstrate cytotoxic potential restricted by TCR-HLA-DR interaction. (A) Enriched CD4⁺ TILs were cocultured in the indicated conditions and cell culture supernatants were analyzed for granzyme B, IFNg, and TNFa production. (B) Live cell imaging of TIL (unlabeled) and target cells (green) loaded with WT or mutated (Q22R) S100A11 peptide cocultured at indicated effector:target (E:T) ratios for 6 hours. Still image acquired at 4 hours time point with 10:1 E:T. Cleaved caspase 3/7 (red) induction was monitored via imaging at regular 30 min intervals and quantified for target cell count (bottom right) and overlap of target cells and cleaved caspase 3/7 (bottom left). Unl=unloaded targets. (C, D) TCR-T or UT peripheral blood lymphocytes (PBL) were cocultured with B cells loaded with S100A11 peptides and blocking antibodies. Effector molecule production was quantified in cell coculture supernatants (C) and cell surface OX40 and 4-1BB expression was evaluated by flow cytometry (D).

Figure 3

Clonal analysis of neoantigen-specific CD4⁺ TIL indicates persistence and effector profile. (A) TCRb sequencing of the infused TIL product for patient 1 displayed as relative productive frequency. Blue slice is the S100A11^Q22R-specific clone. (B) Relative productive frequency of the S100A11^Q22R-specific clone tracked longitudinally in PBMC samples at the indicated weeks (W) following TIL infusion. (C) Uniform manifold approximation and projection (UMAP) of the S100A11^Q22R-specific CD4⁺ TIL clone (left) and the residual CD4⁺ TIL (rCD4) from Patient 1. Cells were colored by clusters. (D) Relative frequency of the clusters displayed in figure 3C. (E) Scaled average expression of reported genes associated with neoantigen specificity in S100A11^Q22R-specific CD4⁺ TIL and rCD4 TIL. Hierarchical clustering was performed on the selected genes and cell types. See online supplemental table 1 for the number of cells in each cluster. (F) Gene set enrichment analysis (GSEA) using DEG comparing S100A11^Q22R-specific CD4⁺ TIL clone vs rCD4 from patient 1 across clusters. Normalized enrichment scores (NES) for pathway analysis were displayed. Hallmark, canonical pathways (CP) and gene ontogeny (GO) data sets were used. Full analysis in online supplemental materials.

Figure 4

Multiple CD4⁺ TIL clones display distinct neoantigen reactivity. (A) Treatment course and CT images of target lesions (red circles) before and after therapy for patient 2. D=day (relative to TIL infusion), Sx=surgery, TL=target lesion. (B) Individual (upper) and sum (lower) measurements of target lesions by longest diameter for patient 2. Red dotted line indicates a −30% change from the baseline sum. (C) Bulk TIL and DC loaded with the neoantigen peptide pool (PP) or no peptide (NP) were cocultured followed by surface staining of OX40 and 4-1BB for cell sorting. (D) Bulk TIL production of granzyme B (GZMB), IFNg, and TNFa was analyzed in sort coculture supernatants. (E) Analysis of IFNg release by TIL in response to individual neoantigen peptides loaded onto APCs. Gray area were below threshold of reactivity. (F) TIL (unlabeled) and neoantigen-loaded target cells (green) were cocultured for 6 hours in a live cell imaging assay to capture cleaved caspase 3/7 (red) induction. Representative image displayed at 4 hours of coculture with 10:1 E:T. (G) Real-time cell-analysis (RTCA) of IFNg-pretreated autologous tumor (ATγ) following addition of neoantigen-specific CD4⁺ TIL. (H) APCs were loaded with neoantigen peptides, precoated with HLA-DR or HLA-DP blocking antibodies, and cocultured with neoantigen-specific CD4⁺ TIL. IFNg release was quantified by the Ella system for each condition. (I) Clonal tracking by TCRb sequencing of neoantigen-specific CD4⁺ TIL clones in the infused TIL product and PBMC at the indicated weeks (W).(J) Distribution of scRNASeq cell clusters present in neoantigen-specific CD4⁺ TIL clones, identified by TCRb sequence. PBMC, peripheral blood mononuclear cell.

Figure 5

TIL product from non-responders contains neoantigen-specific CD4⁺ T cells. (A, F) Sorted neoantigen-specific TIL from patient 3 (A) and patient 4 (F) were cocultured with autologous B cells loaded with neoantigen peptide pool (PP) or no peptide (NP). OX40 and 4-1BB induction was analyzed by flow cytometry on the indicated T cell populations. B, G. Granzyme B (GZMB), IFNg, and TNFa were quantified in coculture supernatants from patient 3 (B) and patient 4 (G). C, H. Sorted TIL were validated for individual neoantigen peptide recognition by IFNg release following coculture with peptide-loaded autologous B cells, respectively, from patient 3 (C) and patient 4 (H). (D, I) Neoantigen peptide hits were loaded on autologous B cells, pretreated with the indicated blocking antibodies, then cocultured with neoantigen-specific CD4⁺ TIL to assess IFNg production from patient 3 (D) and patient 4 (I). (E) Effector molecule secretion and OX40 and 4-1BB induction on neoantigen-specific CD4⁺ TIL from patient 3 were quantified in response to AT-CIITA loaded with neoantigen peptide hits. (J) Clonal frequency of neoantigen-specific CD4⁺ TIL from patient 4 in the infused TIL product and PBMC at the indicated weeks (W) following ACT. (I) Frequency of the scRNASeq cell clusters presents within neoantigen-specific CD4⁺ TIL clones from patient 4. ACT, adoptive cell therapy; PBMC, peripheral blood mononuclear cell.