Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression
Regina Lin, Ling Chen, Gang Chen, Chunyan Hu, Shan Jiang, Jose Sevilla, Ying Wan, John H. Sampson, Bo Zhu, Qi-Jing Li
Regina Lin, Ling Chen, Gang Chen, Chunyan Hu, Shan Jiang, Jose Sevilla, Ying Wan, John H. Sampson, Bo Zhu, Qi-Jing Li
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Research Article Oncology

Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression

Abstract

CD8+ cytotoxic T lymphocytes (CTLs) have potent antitumor activity and therefore are leading candidates for use in tumor immunotherapy. The application of CTLs for clinical use has been limited by the susceptibility of ex vivo–expanded CTLs to become dysfunctional in response to immunosuppressive microenvironments. Here, we developed a microRNA-targeting (miRNA-targeting) approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor BLIMP-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CTLs, and expression correlated with impaired antitumor potential of patient CTLs. We determined that tumor-derived TGF-β directly suppresses CTL immune function by elevating miR-23a and downregulating BLIMP-1. Functional blocking of miR-23a in human CTLs enhanced granzyme B expression, and in mice with established tumors, immunotherapy with just a small number of tumor-specific CTLs in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CTLs that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-β–mediated tumor immune-evasion pathway.

Authors

Regina Lin, Ling Chen, Gang Chen, Chunyan Hu, Shan Jiang, Jose Sevilla, Ying Wan, John H. Sampson, Bo Zhu, Qi-Jing Li

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Figure 3

Functional blockade of miR-23a enhances CTL effector responses ex vivo.

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Functional blockade of miR-23a enhances CTL effector responses ex vivo. ...
(A–C) Purified naive pMel-1 CTLs were activated with anti-CD3/anti-CD28 for 48 hours in vitro, with or without 50 nM FAM-tagged anti–miR-23a LNA. (A) Gating strategy to identify CTLs that have taken up the anti–miR-23a LNA. (B) CTL effector molecule expression in untreated (0 nM LNA), FAM– LNA-treated (50 nM LNA; FAM–), and FAM+ LNA-treated (50 nM LNA; FAM+) CTLs. Representative histograms of n = 6 independent experiments. (C) MFI of CTL effector molecules in FAM– and FAM+ CTLs treated with 50 nM anti–miR-23a LNA; n = 7. P values were determined by 2-tailed paired t test. (D) Schematic of the mock decoy and miR-23a decoy retroviral expression vectors. iRFP was the internal marker for monitoring transfection efficiency; puromycin resistance (PuroR) was the selection marker for enriching engineered cells; GFP was a reporter for miR-23a sequestration and decoy function. The iRFP and GFP decoy expression cassettes were separated by an insulator (51) (black oval). (E) Left: Representative dot plots of iRFP and GFP expression in pMel-1 CTLs transduced with the miR-23a decoy, where iRFP was used as the internal marker. Right: Quenching of GFP intensity by the miR-23a decoy in CD8+iRFP+ CTLs. Bar graph represents mean ± SEM; n = 6. (F) pMel-1 CTLs retrovirally transduced with a mock decoy vector or the miR-23a decoy vector were assessed for CTL effector molecule expression in vitro. P values were determined by 2-tailed paired t test. (G) In vitro cytotoxicity of sorted iRFP+ mock and miR-23a decoy–expressing OT-I CTLs. Representative data of n = 3 independent experiments.