April 30, 2022

Cytotoxic CD8+ T cells (CTLs) are a crucial part of the immune system known for their ability to eliminate rapidly multiplying malignant cells. Until the publication of a Mayo Clinic study in Cancer Immunology Research in February 2022, the T-cell intrinsic factors required for human CTLs to be highly effective in killing tumor cells were not well defined.

The study evaluated CTLs based on patient response to treatment with immune checkpoint inhibitors. By analyzing the activity of both nonresponders and responders to treatment, the researchers aimed to identify key factors associated with effective CTL function.

"Most patients with advanced cancers have not benefited from current immune checkpoint blockade therapy," said Haidong Dong, M.D., Ph.D., an immunology researcher at Mayo Clinic in Rochester, Minnesota. "Our study provides a tool to detect this problem and also provides an mRNA-based therapy to fix it."

The study found that transfecting the CTLs with mRNA of a certain gene involved in tumor killing improved response to immunotherapy for those who previously had little response to the therapy.

Effective immunotherapy for a small number of patients

This study reviewed anti-PD-1 and anti-PD-L1 immunotherapies that are based on the discovery of PD-L1 (also named B7-H1) by Dr. Dong and a former Mayo Clinic research colleague between 1998 and 2002. These treatments are among the most widely used of cancer immunotherapies and can lead to complete remission, but only in a small proportion of patients. The majority of people treated with anti-PD-1 and anti-PD-L1 experience no detectable response.

Initially, this treatment was thought to be effective because it rescued exhausted T cells in the tumor microenvironment. However, recent research has indicated that expansion and circulation of peripheral CTLs are important because these cells infiltrate tumors and are responsible for tumor killing.

Analyzing CTL expression

In the Mayo Clinic study, the researchers allocated samples randomly whenever possible and no data were excluded from the analysis. Researchers took the following steps to identify the differences in gene expression within the CTLs of patients who did and did not respond to treatment.

• Performed single-cell RNA-sequencing analysis of 16 paired patient samples taken before and after initiation of immunotherapy
• Determined that reduced natural killer cell granule protein-7 (NKG7) was a common feature among nonresponders and validated this finding in separate sets of patient data
• Characterized the function of NKG7 in human CTLs via knockdown of NKG7 in primary cells followed by cytotoxicity assays, time-lapse imaging studies, electron microscopy and calcium-flux measurements
• Studied the effect of overexpression of NKG7 through transfection with NKG7 mRNA both in human samples taken from nonresponding patients and in antigen-specific CTLs from mice
• Screened for transcription factors that modulate NKG7 gene expression through small interfering RNA-mediated knockdown of transcription factors followed by chromatin immunoprecipitation quantitative polymerase chain reaction (PCR), quantitative real-time PCR, Western blot and functional cytotoxicity assays

For the human samples analyzed, the researchers retrospectively collected clinical course, treatment information and outcomes of treatment with anti-PD-1 and anti-PD-L1 at Mayo Clinic. Outcomes of treatment were evaluated using standard clinical practice guidelines and the Response Evaluation Criteria in Solid Tumors (RECIST) methodology.

mRNA transfection for improved tumor killing

Single-cell RNA-sequencing analysis of peripheral CTLs from patients treated with anti-PD-1 therapy indicated that cells from nonresponders did not show as much of the cytolytic granule-associated molecule NKG7. Reduced NKG7 expression altered cytolytic granule number, trafficking and calcium release, resulting in decreased killing of tumor cells by CTLs.

Transfection of CTLs with NKG7 mRNA improved tumor-cell killing ability and increased response to anti-PD-1 or anti-PD-L1 in vitro. NKG7 mRNA therapy also improved the response of antigen-specific CTLs in mice in an in vivo model of adoptive T cell therapy.

"We found that by introducing mRNA in immune cells, it is possible to produce useful proteins to improve anti-tumor activity without attempting to change the genome itself," said Dr. Dong. "This approach may have the potential to be used across the spectrum of medicine to pull information gained from single-cell RNA-sequencing into mRNA-based therapy for patients."

The study also showed that transcription factor ETS1 played a role in regulating the activity of NKG7. All of these results show that NKG7 is a necessary component of the tumor response for CTLs and is a T cell intrinsic therapeutic target for enhancing cancer immunotherapy.

For more information

Wen T, et al. NKG7 is a T-cell-intrinsic therapeutic target for improving antitumor cytotoxicity and cancer immunotherapy. Cancer Immunology Research. 2022;10(2):162.

Refer a patient to Mayo Clinic.