citations

                    Targeting P4HA1 promotes CD8+ T cell progenitor expansion toward immune memory and systemic anti-tumor immunity
                

Authors:

Ma S, Ong LT, Jiang Z, Lee WC, Lee PL, Yusuf M, Ditzel HJ, Wang Y, Chen Q, Wang W, Wu X, Tan EY, Yu Q.

In:

Source: Cancer Cell
Publication Date: (2025)
Issue: S1535: 6108(24)00476-8

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Gene Expression

Basic Research

Molecular Biology

Regenerative medicine

Drug Discovery

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Generation of gene knockout CAR T cells

Gene knockout CAR T cells were generated using the 4D-Nucleofector X kit S (Lonza, Switzerland), according to the manufacturer’s instructions. Briefly, the gRNA and SpCas9 nuclease (Integrated DNA Technologies IDT) were mixed at a molar ratio of 3:1 for 20 minutes to form RNP complex under room temperature. CAR T cells were washed with 1x PBS for three times and resuspended in Nucleofector Solution. RNP complex was then mixed with CAR T cells at 150 pmol gRNA per million cells in 20 µL solution and subjected to the 4D-Nucleofector X Unit (Lonza) for electroporation under the program EO-115. Post electroporation, cells were transferred to pre-warmed ImmunoCult-XF medium for recovery and released to a fresh complete medium containing IL-7 and IL-15 for further analysis after 4 hours. Sequences of gRNA were summarized in Key Resource Table and synthesized by Integrated DNA Technologies IDT.

Abstract

Successful immunotherapy relies on both intratumoral and systemic immunity, which is yet to be achieved for most patients with cancer. Here, we identify P4HA1, encoding prolyl 4-hydroxylase 1, as a crucial regulator of CD8+ T cell differentiation strongly upregulated in tumor-draining lymph nodes (TDLNs) and hypoxic tumor microenvironment. P4HA1 accumulates in mitochondria, disrupting the tricarboxylic acid (TCA) cycle through aberrant a-ketoglutarate and succinate metabolism, promoting mitochondria unfitness and exhaustion while suppressing progenitor expansion. Targeting P4HA1 enhances both adoptive and endogenous TCF1+ CD8+ T progenitor expansion while mitigating the development of exhaustion in the tumor, TDLN, and blood, enabling a notable and durable systemic anti-cancer immunity. We propose that P4HA1 induction in CD8+ T cells in cancer orchestrates an immune-escape program, offering a T cell-directed target for system immunotherapy in solid tumors.

Open in PubMed