Journal: Cell Reports Medicine

Article Title: Lymphodepleting chemotherapy potentiates neoantigen-directed T cell therapy by enhancing antigen presentation

doi: 10.1016/j.xcrm.2025.102506

Figure Lengend Snippet: Identification of a sensitive and specific TCR targeting KRAS.G12V-A∗03:01 (A) Estimation of the prevalence of KRAS.G12V and HLA-A∗03:01 across various cancers using TCGA and CBI portal data. Data are presented as the percentage of patients estimated in this dataset. (B) HLA immunopeptidomics panel for RAS.G12V mutation, including quantification of VVVGAVGVK peptide via high-sensitivity targeted mass spectrometry. Data shown are from one biological replicate. (C) Representative flow cytometry plots of CD8 4-1BB activation (left) and IFN-γ secretion (right) from healthy donors (D98 and D104) post stimulation with mutated KRAS.G12V or WT peptides. Numbers indicate the percentage of gated events. Data are representative of two independent biological replicates, each with three technical replicates. (D) Representative flow cytometry plots showing KRAS.G12V-HLA-A∗03:01 dextramer staining of a pancreatic cancer patient that harbors the KRAS.G12V mutation and HLA-A∗03:01 (left) PBMCs and a pancreatic cancer patient without the HLA-A3 allele (right). Numbers indicate the percentage of CD8 + double-dextramer + cells. Data are representative of two independent biological replicates, each with three technical replicates. (E) Representative flow cytometry plots showing KRAS.G12V dextramer staining of D104 T cells stimulated against the WT KRAS peptide (left) or the KRAS.G12V peptide (right) in vitro . Data are representative of two independent biological replicates, each with three technical replicates. (F) Single-cell TCR sequencing clone distribution. Dextramer-double positive clones are highlighted in blue and double-negatives in gray. Data shown are from one biological replicate. (G) The table includes the sequence of the major TCR clone identified in (F). (H) Flow cytometry quantification of IFN-γ (left) and TNF-α (right)-positive TCR-T104 cells after co-culturing with KRAS.G12V or WT-pulsed B cells. Gated population from CD8 + mTCR + . Graphs represent mean ± SEM. The p value is calculated by the Mann-Whitney U test. Data are representative of five independent biological replicates, each with three technical replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (I) Flow cytometry quantification showing %4-1BB-positive TCR-T104 cells after incubation with pulsed B cells across a serial dilution of mutant or WT peptides. Data are representative of two independent biological replicates, each with three technical replicates. Each dot represents the mean of the technical replicate. The line color depicts the KRAS epitope that was pulsed (WT or mutant). (J) Luminescence mean fold change ± SEM of CD8 + Jurkat cells expressing T104-TCR across serial dilutions of WT or G12V mutant peptides pulsed on B cells. Data are representative of two independent biological replicates, each with three technical replicates. (K) Flow cytometry quantification showing %4-1BB-positive cells of TCR-T104 T cells co-cultured with endogenous lung cancer cell lines expressing the KRAS.G12V mutation (H441) or not (H3122, left), and colon cancer line (SW620) with or without A∗03:01 (right). Graphs represent mean ± SEM. The p value is calculated by the Mann-Whitney U test. Data are representative of two independent biological replicates, each with three technical replicates. (L) Flow cytometry quantification showing mean fold change ± SEM of cleaved caspase-3-mediated killing assay, using T104-TCR-T cells or irrelevant TCR (17.1.2) co-cultured with either KRAS.G12V-expressing lung cancer cell line (H441) or KRAS.Q61K cell line (Calu6). Gating on far-red-labeled+ cancer cells. The p value is calculated by the ANOVA test followed by Tukey HSD multiple correction test. Data are representative of two independent biological replicates, each with three technical replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (M) Killing dynamics of KRAS.G12V-specific T104-TCR-T cells (left), or irrelevant TCR (17.1.2 TCR, right) co-cultured with SW620-A∗03:01-GFP. GFP-positive area was normalized to time zero. Data are presented as mean ± SEM. Data are representative of four independent biological replicates, each with three technical replicates. The last time point was tested by a two-sided Student’s t test and was significant in all effector-to-target (E:T) ratios for T104 but not for irrelevant TCR ( p < 0.05). (N) Flow cytometry quantification of IFN-γ (left), TNF-α (middle), and 4-1BB (right) comparing three TCRs against 1 μg KRAS.G12V/WT-pulsed HLA-A∗03:01 B cells. Data are presented as mean ± SEM, and the p value is calculated by two-way ANOVA followed by Šidák correction for multiple comparisons. Data are representative of three independent biological replicates, each with three technical replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (O) Killing dynamics comparing three TCRs co-cultured with SW620-A∗03:01-GFP (left) and SW620-GFP (right). E:T ratio shown was maintained at 2:1 for all TCRs, normalized to the transduction rates of mTCR + cells. GFP-positive area was normalized to time zero. Data are presented as mean ± SEM. Data are representative of two independent biological replicates, each with three technical replicates. See also .

Article Snippet: The Calu6 and H441 lung cancer cell-lines were purchased from ATCC.

Techniques: Immunopeptidomics, Mutagenesis, Mass Spectrometry, Flow Cytometry, Activation Assay, Staining, In Vitro, Sequencing, Clone Assay, MANN-WHITNEY, Incubation, Serial Dilution, Expressing, Cell Culture, Labeling, Transduction