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920 ETS-1 regulates the activation of human CD8 T-Cells in response to immune checkpoint inhibitors
  1. Jacob Hirdler1,
  2. Zhiming Mao2,
  3. Cristina Correia1,
  4. Fabrice Lucien-Matteoni1 and
  5. Haidong Dong3
  1. 1Mayo Clinic, Rochester, MN, USA
  2. 2Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, USA
  3. 3Mayo Clinic College of Medicine, Rochester, MN, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background It is estimated approximately ~12% of checkpoint blockade recipients respond to treatment. We have previously identified NKG7 as a therapeutic target that is upregulated in CD8 T cells of responders and now ETS-1(Ets Proto-oncogene 1) as an upstream modifier of its expression. With a focus on mRNA therapeutics, we have identified ETS-1 as a modulation candidate capable of increasing response to immunotherapy. ETS-1 in hematopoietic stem cell development has been extensively researched, yet its role in mature CD8 T cell activation and persistence is still lacking. By combining RNAseq, Western blot, and cytotoxicity assay data from human CD8 T cells derived from healthy donors we identified the transcription factor ETS-1 plays an important role in a checkpoint-dependent (PD-1/PD-L1) adaptive immune response.

Methods Utilizing in silico analysis and transcription factor binding motif prediction software (PROMO) we identified ETS-1 binding motif upstream of NKG7. From here we nucleofected ETS-1 siRNA in human primary CD8 T cells to observe the consequence on NKG7 protein expression via Western blot. From here we validated that ETS1 functions as a negative regulator for expression of NKG7 and Granzyme B mRNA via RT-qPCR and Western blot in resting CD8 T cells. Next, we submitted ETS-1 knockdown samples for bulk RNAseq and differential gene expression (DEG) analysis (3 control/3 siETS1). (adj p-value <0.05 and |Log2 FC| >0.5). Lastly, for a functional readout, we utilized our calcein-release cytotoxicity assay. We cocultured control and siETS-1 CD8 T cells with/without Pembrolizumab (anti-PD-1) with calcein-labelled target cells (MCF7) quantifying cytotoxic tumor killing.

Results Knockdown (KD) of ETS-1 in resting human primary CD8 T cells resulted in higher expression of NKG7 in 6 of 7 RT-qPCR samples and 3 of 3 Western blot assays (p value 0.11). DEG analysis of CD8 T cells revealed 123 genes differentially expressed in response to KD of ETS-1. GO pathway enrichment analysis showed significant association of ETS-1 KD with T cell activation. In line with this, ETS1 KD in CD8 T cells resulted in higher cytotoxicity for 9 of 10 healthy donors (median control 28.2%/ siETS1 36.7% p value = .0184). Lastly, ETS-1 KD led to increase of T-cell cytotoxicity for 2 of 3 donors in response to PD-1/PD-L1 checkpoint blockade in vitro.

Conclusions We demonstrate the selective knockdown of ETS-1 in primary CD8 T cells not only increases cytotoxicity but also displays a potential avenue for increasing the response rate of immunotherapy via gene expression augmentation.

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