Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells
Authors: Ripamonti Chiara , Spadotto Valeria , Steinkuhler Christian , Fossati Gianluca 
Published: March 2024

Abstract
Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I.
Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4586.