Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients

Abstract

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4⁺, and CD8⁺ memory T cells targeting the mutated KRAS^G12D and KRAS^G12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8⁺ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.

Fig. 1

Identification of neoantigen-specific T cells in PBMC of epithelial cancer patients. a A representative data from one of four patients showing the gating strategy used for the phenotypic analysis of PD-1-positive (PD-1⁺) peripheral T cells. b PBMC isolated from four epithelial cancer patients were thawed, rested overnight in a cytokine-free T-cell medium and stained with antibodies against CD3, CD45RO, CD62L, and PD-1. PD-1⁺ and PD-1-negative (PD-1⁻) cells were analyzed for the surface expression of the T-cell memory markers, CD45RO and CD62L. Error bars indicate the mean standard deviation between all four patients samples. c PBMC isolated from six epithelial cancer patients were thawed, rested overnight in a cytokine-free T-cell medium and stained with antibodies against CD3, CD45RO, CD62L, CCR7, and CD45RA. Cells were sorted based on their memory subset, washed once with PBS, frozen at −80 °C and send for TCR-Vβ sequencing. Numbers represent the percent of specific TCR-Vβ sequence in each T-cell population. A minus sign (−) was used If no sequence was identified. d Analysis of the productive clonality of TCR-Vβ sequences of each T-cell population isolated in c. (Paired t-test, *** P < 0.001, * P < 0.05) dashed line, mean

Fig. 2

Identification of T cells specific for mut SMAD5. a Memory and naive CD8 cells were co-cultured with autologous DCs transfected with 13 TMGs harboring 201 mutated 25mer sequences for 18 h. Activated T Cells were stained for CD3, CD8, 41BB, and OX40 and sorted based on 41BB and OX40 expression to enrich for neoantigen-reactive cells. b Memory, T_N and PBMC were co-cultured with autologous DCs transfected with TMGs 1–13 for 18 h, stained with CD3, CD8, and 41BB and analyzed for surface expression of 41BB as a marker for T-cell activation. c Memory CD8 cells isolated in A were co-cultured for 18 h with autologous DCs that were individually pulsed with the mutated peptides encoded by TMG-8 and tested either by flow cytometry for 41BB expression or IFNγ-secretion using ELISPOT assay. d Naive CD8 cells isolated in a were co-cultured for 18 h with autologous DCs that were individually pulsed with the mutated peptides encoded by TMG-6 and tested either by flow cytometry for 41BB expression or IFNγ-secretion using ELISPOT assay. e Memory CD8 cells isolated in a were co-cultured for 18 h with autologous DCs that were loaded with WT or Mut SMAD5 LP, predicted minimal epitope (ME) and full-length WT and mutated SMAD5 RNA. Cells were tested for antigen recognition by flow cytometry for 41BB expression or IFNγ-secretion using ELISPOT assay. f TCR-transduced PBLs were co-cultured with DCs pulsed with a serial dilution of SMAD5 Mutated or WT peptides. g COS7 cells were transfected with patient’s class I HLA and co-cultured with TCR-transduced cells. Reactivity was determined by ELISPOT for IFNγ

Fig. 3

Identification, isolation, and analysis of KRAS^G12V-reactive TCR. a 41BB-enriched CD8 memory T cells were expanded, and their reactivity was tested against autologous DCs pulsed with the indicated peptides. b 41BB-enriched CD8 memory T cells are stained with KRAS^G12V 9mer tetramers; unstimulated CD8 cells were used as control (left). c 41BB upregulation and ELISPOT IFNγ secretion assay of TCR-transduced allogeneic T cells. T cells co-cultured with cell lines naturally expressing G12 mutations ± HLA-A11 transduction. d IFNγ secretion of TCR-transduced cells against autologous DCs pulsed with the indicated concentrations of mutated and WT 9mers. A representative of at least three experiments

Fig. 4

Isolation of HLA-DRB1*08:01-restricted TCR-targeting KRAS^G12D. a IFNγ-ELISPOT assay of 41BB⁺ and/or OX40⁺ enriched CD4 subsets co-cultured with autologous DCs pulsed with mutated and wild-type KRAS peptides. b TRBV sequences of 41BB⁺-sorted T cells following co-culture with DCs pulsed with KRAS^G12D peptide. c–e Retrovirally transduced allogeneic PBLs expressing the indicated TCRs were further tested. c Transduced PBLs were co-cultured with autologous DCs pulsed with either KRAS^G12D or KRAS^WT. d TCR1-transduced PBLs were co-cultured with DCs pulsed with a serial dilution of G12D or WT peptides. e COS7 cells were transfected with patient’s class II HLA and co-cultured with TCR1-transduced cells. Reactivity was determined by IFNγ ELISA and the upregulation of 41BB surface marker. c–e are representative of at least two experiments