2025 American Academy of Neurology Abstract Website

Objective:

To study the role of NK cells in antitumoral effects of replicating retrovirus expressing IL-15 fusion protein RLI.

Background:

Glioblastoma (GBM) is a highly aggressive brain tumor that is the most common and lethal primary brain tumor in adult. Understanding that IL-15 is an immunogenic cytokine that is known to stimulate T-cell activation and proliferation, we have successfully delivered an IL-15 superagonist (RLI) through a replicating retrovirus RRV-RLI, leading to an increased survival in tumor-bearing mice and an increased level of intratumoral T-cells.

Design/Methods:

We harvested three spleens from the C57/BL6 mice after euthanasia. The spleens were processed and used to isolate NK cells using negative selection method through commercially available Mojosort Mouse NK isolation kit. Cell count was checked to maintain the optimal concentration of isolated NK cells. Following that, three SB28 cell lines (RRV-RLI, RRV-vector-only (RRV), and wild type (WT)) were used to co-culture NK cells at 5:1 and 10:1 effector-to-target (E-to-T) ratios for 24-hour and 48-hour co-culture time periods. The co-cultured wells (three cell lines and two ratios) were collected in the 24-hour and 48-hour timepoints. Compensation beads and FMOs were prepared for day 1 to run along with the 24-hour group wells. FlowJo was used to analyze the results.

Results:

There was a significantly greater presence of NK cells in the SB28 RRV-RLI cells at the 48-hour timepoint. Within the NK1.1 gated cells, there was a significantly higher co-expression of CD69 and Granzyme B for the RRV-RLI cohort compared to the other two cohorts. Individual expressions of Granzyme B and CD69 were also significantly increased in RRV-RLI cohorts.

Conclusions:

In conclusion, the presence of NK cells as well as proliferation and cytotoxicity were shown to be higher in RRV-RLI cohort in comparison to the RRV or WT cohorts.