Miltefosine Reinvigorates Exhausted T Cells by Targeting Their Bioenergetic State

Academic Background

T cell exhaustion is a significant challenge in immunotherapy, particularly in cancer treatment. It typically occurs when T cells are chronically exposed to antigen stimulation, leading to a gradual loss of function, characterized by reduced effector function, increased expression of inhibitory receptors, altered epigenetic profiles, decreased cytokine production, impaired proliferation, and suppressed mitochondrial respiration and glycolysis. This phenomenon was first identified in the chronic lymphocytic choriomeningitis virus (LCMV) infection mouse model but has since been found to be prevalent in various diseases, especially malignancies. Exhausted CD8+ T cells are heterogeneous in phenotype and function, with progenitor-exhausted T cells retaining some proliferative capacity and responding to anti-PD-1 checkpoint blockade, while terminally exhausted T cells lose their proliferative ability and do not respond to existing immunotherapies.

Despite significant progress in treating certain cancers with immune checkpoint inhibitors (ICBs) and chimeric antigen receptor T cell (CAR-T) immunotherapy, T cell exhaustion remains a major barrier to efficacy. Therefore, identifying drugs or strategies to reverse T cell exhaustion is of great clinical importance.

Source of the Paper

This paper was co-authored by Xingying Zhang, Chenze Zhang, Shan Lu, and others, with the research team coming from multiple institutions including the Institute of Zoology, Chinese Academy of Sciences, Beijing University of Chinese Medicine, and the Chinese People’s Liberation Army General Hospital. The paper was published on December 17, 2024, in Cell Reports Medicine, titled “Miltefosine Reinvigorates Exhausted T Cells by Targeting Their Bioenergetic State.”

Research Process

1. Generation of Hypofunctional CAR-T Cell Model

To mimic the T cell exhaustion state, the research team generated hypofunctional CAR-T cells through multiple rounds of tumor cell stimulation. The specific steps included: - Co-culturing CAR-T cells (M28Z) with a mesothelin-expressing lung cancer cell line (NCI-H226-luciferase) at a 1:1 ratio. - Through multiple rounds of stimulation, the CAR-T cells gradually lost their tumor-killing ability, exhibiting exhaustion characteristics. - RNA sequencing (RNA-seq) and flow cytometry analysis confirmed the exhaustion phenotype of these CAR-T cells.

2. Screening of FDA-Approved Compound Library

The research team established a high-throughput drug screening platform based on hypofunctional CAR-T cells, screening 1700 FDA-approved small molecule compounds. The screening criterion was the ability to enhance the tumor-killing ability of CAR-T cells. Through primary and secondary screening, miltefosine was identified as a candidate drug.

3. Effects of Miltefosine on CAR-T Cell Function

Miltefosine, an antiparasitic drug, was found to significantly enhance the tumor-killing ability of hypofunctional CAR-T cells. Further experiments showed that miltefosine restored the impaired glycolysis and oxidative phosphorylation metabolism in CAR-T cells, thereby improving their function.

4. Single-Cell RNA Sequencing Analysis

Through single-cell RNA sequencing (scRNA-seq), the research team found that the proportion of effector cells significantly increased in miltefosine-treated CAR-T cells, while the proportion of exhausted cells decreased. This indicates that miltefosine can shift CAR-T cells from an exhausted state to a functional state.

5. Mechanism of Action of Miltefosine

Miltefosine enhances the glycolysis and glucose uptake capacity of CAR-T cells, restoring their metabolic function. Further research showed that the effect of miltefosine depends on the glucose transporter GLUT1. Pharmacological and gene knockout experiments validated the key role of GLUT1 in miltefosine’s enhancement of CAR-T cell function.

6. In Vivo Validation

In cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models, miltefosine significantly enhanced the antitumor efficacy of CAR-T cells. Particularly in the terminally exhausted state, miltefosine was still able to improve CAR-T cell activity, whereas anti-PD-1 antibodies were ineffective.

Main Results

1. Generation of Hypofunctional CAR-T Cell Model: Through multiple rounds of tumor cell stimulation, CAR-T cells with exhaustion characteristics were successfully generated, and their exhaustion phenotype was validated through RNA-seq and flow cytometry.
2. Identification of Miltefosine: Through high-throughput screening, miltefosine was identified as a candidate drug that significantly enhances the tumor-killing ability of hypofunctional CAR-T cells.
3. Miltefosine Restores CAR-T Cell Function: Miltefosine restored the impaired glycolysis and oxidative phosphorylation metabolism in CAR-T cells, thereby improving their function.
4. Single-Cell RNA Sequencing Reveals Population Shift: In miltefosine-treated CAR-T cells, the proportion of effector cells significantly increased, while the proportion of exhausted cells decreased.
5. Mechanism of Action of Miltefosine: Miltefosine enhances the glycolysis and glucose uptake capacity of CAR-T cells, restoring their metabolic function, and this effect depends on GLUT1.
6. In Vivo Validation: In CDX and PDX mouse models, miltefosine significantly enhanced the antitumor efficacy of CAR-T cells, particularly in the terminally exhausted state.

Conclusion

This study established a hypofunctional CAR-T cell model and identified miltefosine as a candidate drug capable of reversing T cell exhaustion. Miltefosine restored the metabolic function of CAR-T cells, significantly enhancing their antitumor efficacy. This discovery provides a new strategy for overcoming T cell exhaustion and improving the efficacy of CAR-T cell immunotherapy.

Research Highlights

1. Innovative Model: The research team successfully generated a hypofunctional CAR-T cell model through multiple rounds of tumor cell stimulation, providing a reliable experimental platform for studying T cell exhaustion.
2. High-Throughput Screening: Through high-throughput screening, miltefosine was identified as a candidate drug capable of reversing T cell exhaustion, offering a new drug option for immunotherapy.
3. Metabolic Mechanism Study: Miltefosine restored the impaired glycolysis and oxidative phosphorylation metabolism in CAR-T cells, highlighting the important role of metabolic regulation in T cell exhaustion.
4. In Vivo Validation: In CDX and PDX mouse models, miltefosine significantly enhanced the antitumor efficacy of CAR-T cells, particularly in the terminally exhausted state, demonstrating its clinical application potential.

Research Significance

This study not only provides new insights into the mechanisms of T cell exhaustion but also offers important experimental evidence for developing drugs to reverse T cell exhaustion. As an FDA-approved drug, miltefosine has promising clinical application prospects and may be used in the future to improve the efficacy of CAR-T cell immunotherapy, especially in treating solid tumors.