Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion
Jacob S. Bowers, Michelle H. Nelson, Kinga Majchrzak, Stefanie R. Bailey, Baerbel Rohrer, Andrew D.M. Kaiser, Carl Atkinson, Luca Gattinoni, Chrystal M. Paulos
Jacob S. Bowers, Michelle H. Nelson, Kinga Majchrzak, Stefanie R. Bailey, Baerbel Rohrer, Andrew D.M. Kaiser, Carl Atkinson, Luca Gattinoni, Chrystal M. Paulos
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Research Article Immunology Oncology

Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion

Abstract

Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell’s therapeutic fitness. In contrast, we discovered that IL-17–producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor–positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence.

Authors

Jacob S. Bowers, Michelle H. Nelson, Kinga Majchrzak, Stefanie R. Bailey, Baerbel Rohrer, Andrew D.M. Kaiser, Carl Atkinson, Luca Gattinoni, Chrystal M. Paulos

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Figure 3

Th17 cells expanded for 3 weeks enter quiescence but retain antitumor potency and ability to engraft.

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Th17 cells expanded for 3 weeks enter quiescence but retain antitumor po...
(A) Growth of Th17 cells in culture; n = 7 independent cultures. (B) CD44 and CD62L memory marker expression on Vβ14+CD4+ T cells before polarization and Th17 cells at different time points in culture analyzed via flow cytometry; representative of 3–5 cultures. (C) Western blot analysis of nuclear protein from 7-, 14-, and 21-day-expanded Th17 cells, T, Tcf7; H, histone H3; representative of 4 independent cultures. (D) Quantified Tcf7 protein relative to histone H3 (mean ± SEM); n = 4 independent cultures. RR (OD), relative ratio of optical density. (E) Mean fluorescence intensities (MFIs) (± SEM) of cytokine and costimulatory receptors during ex vivo culture of Th17 cells assessed by flow cytometry; n = 5 independent cultures. (F) Mice with B16F10 tumors were treated with equal numbers (2 × 106 cells/mouse) of Th17 cells from the early, intermediate, and late time points of ex vivo culture following 5 Gy total body irradiation 1 day prior; n = 8–9 mice/group, representative of 4 independent experiments. (G) Percentage survival of mice treated in Figure 3F; n = 8–9 mice/group, Kaplan-Meier curves compared by log-rank test. (H) Mean number (± SEM) of donor Th17 cells from early or late culture time points in host C57BL/6 spleen, tumor, and eye at indicated days after treatment; n = 4–5 mice, representative of 2 independent experiments. Mean cell numbers of early or late Th17 cells compared by Student’s t test. TRP, CD4+ T cells with a TCR specific for tyrosinase-related protein-1. (I) Hybridized telomere peptide nucleic acid (PNA) in Th17 cells at early and late culture time points; n = 3 independent cultures. (J) MFI (± SEM) of telomere length in donor Th17 cells 4 weeks after treatment; n = 5 mice/group. Telomere MFIs compared by Student’s t test. **P = 0.0021. ns, not significant.