p53 Isoform D133p53a: A Novel Transcriptional Enhancer of T-Cell Effector Function to Improve T-Cell Based Cancer Immunotherapy

Background:

Adoptive transfer of genetically modified T lymphocytes with tumor antigen-specific receptor has proven efficacy in cancer immunotherapy. However, in many patients the overall benefit is still limited due to various tumor escape mechanisms. Cell damage and metabolic/hypoxic stress in the tumor microenvironment (TME) can lead to a dysfunctional anti-tumor T cell response called T cell senescence. The tumor suppressor TP53 is a master molecule in the regulation of cell cycle and senescence. Few studies have demonstrated the critical role of p53 isoforms in the regulation of cellular senescence mainly in tumor cells. However, their role in tumor infiltrating lymphocytes (TILs) remains largely unexplored.

Aims:

Strategies to prevent T cell senescence in the TME could improve T cell function and thus anti-tumor response. To better understand the role of D133p53 isoform in regulating the cell cycle and senescence we studied the cellular and metabolic/energetic phenotype as well as the effector function of the D133p53-modified tumor-antigen (TA) specific human T cells. We further aimed at identifying the mechanism that may regulate this phenotype.

Methods:

T cells form healthy donors were retrovirally co-transduced with a TA-specific T cell receptor (TCR) together with the D133p53 isoform or an empty control vector. Modified T cells were characterized for the expression of key activating/inhibitory molecules, homing markers and for their proliferation capacity by flow cytometry. Additionally, we determined the metabolic and energetic phenotype of the cells with an Agilent Seahorse XFp Analyzer. The effector functions i.e. cytokine secretion and antigen-specific killing capacity were assessed by Luminex immunoassay and long-term tumor colony-forming assay, respectively. In an attempt to identify molecules/pathway contributing to this phenotype we performed quantitative proteomic-based analysis.

Results:

Our analyses of human T cells simultaneously engineered with D133p53a-isoform and a TA-specific TCR revealed reduced cell surface expression of T-cell inhibitory molecules (i.e. PD-1 or TIGIT), senescence markers (CD57, CD160) and increased expression of the homing receptor CD62L upon TA stimulation. First comparative analyses between D133p53a-modified and control T-cells revealed changes in the cell's metabolic and energetic program similar to quiescent/naïve T cells. D133p53a-T cells exhibited lower ATP production, oxygen consumption as well as lower glucose utilization. Upon antigen-specific stimulation, however, they increased their metabolic activity up to the levels of control cells. Importantly, while control T cells exhibited replicative senescence after chronic antigen stimulation, D133p53a-expressing T cells remained highly proliferative, showed superior cytokine secretion and enhanced tumor-specific killing capacity. Comparative proteomic analysis revealed significant differences in more than 100 proteins. Detailed pathway and network analysis as well as validation of the most significantly changing proteins is currently performed.

Conclusion:

By providing insights in the regulation of T cell metabolic changes and underlying mechanisms that limit immunosenescence, genetic modification with p53 isoforms could be a promising strategy to circumvent tumor-mediated T cell dysfunction and represents a novel approach with high potential for cancer immunotherapy.