Researchers develop T cell growth method that enhances cancer-fighting ability in melanoma model

By Dr. Priyom Bose, Ph.D.Reviewed by Danielle Ellis, B.Sc.Jan 31 2025

Researchers have developed a new method to grow T cells that have greater longevity and can more efficiently destroy cancer cells in mouse models of melanoma compared to those grown in traditional growth media

A recent Cell Metabolism study assessed how the therapeutic efficacy and metabolism of T cells can be improved by redirecting the glucose flux toward mitochondria using dichloroacetate (DCA) conditioning.

What factors hinder the effectiveness of immunotherapies for cancer treatment?

Immunotherapy plays an important role in treating cancer patients. The effectiveness of the existing therapies could be enhanced through a population of dormant tumor-reactive T cells, which can be re-invigorated.

The mode of action of different cell therapies varies substantially. For example, tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cell therapy (ACT), which is based on the direct proliferation of lymphocytes harvested from the patient’s tumor tissue. In contrast, CAR-T cell therapy and T cell receptor (TCR) transgenic T cell therapy rely on genetic tools to redirect the T cells of the patient to identify tumor-associated antigens.

A common aspect of all forms of ACT is in vitro culture, where mitogenic stimuli and cytokines are used to develop a high number of tumor-reactive T cells for reinfusion. In contrast to liquid tumors, solid tumors are resistant to ACT, possibly because of the negative effects of the tumor microenvironment (TME) on T cell metabolism, function, and persistence.

About the study

The currently available in vitro culture strategies for growing tumor-reactive T cells depend on supraphysiologic levels of glucose and other metabolites. Therefore, there is a considerable metabolic divergence between T cells grown in vitro and in vivo. The current study hypothesized that the hypermetabolic, mostly hyperglycemic, condition used to grow T cells for therapy could generate stress that decreases their therapeutic efficacy.

Several experiments were conducted to test whether the newly developed in vitro protocol enhanced the therapeutic efficacy of T cells for melanoma treatment in mouse models.

Study findings

Experimental findings indicated that activating T cells for 48 hours in combination of certain stimuli could encourage aerobic glycolysis. The highest level of aerobic glycolysis was noted with a combination of three stimuli, namely T cell receptor (TCR) triggering, CD28 costimulation, and hyper-physiologic concentrations of IL-2.

To generate an ‘‘in vivo-activated’’-like state and enhance efficacy, freshly isolated TCR-Tg pmel-1 CD8+ T cells were activated using antigen-presenting cells, cognate peptide (gp100), and IL-2. A 7-day T cell conditioning with DCA reduced extracellular lactate and increased total acetyl-coenzyme A (CoA) by changing glycolytic flux.

Pyruvate dehydrogenase kinase 1 (PDHK1) was inhibited by DCA and DCA-expanded T cells showed a highly varied metabolome, higher mitochondrial mass, and altered mitochondrial physiology. The mitochondria were smaller on average exhibiting denser cristae and greater mitochondrial fusion. These changes resulted in greatly increased spare respiratory capacity. Importantly, at the end of the expansion, DCA-conditioned T cells consumed less glucose but were more efficient at oxidizing glucose.

To assess the effects of DCA conditioning on the potency of therapeutic T cells in cancer immunotherapy, these cells were used to treat mice bearing B16 melanoma. Cytotoxicity assays showed a small but significant improvement in the therapeutic potential of DCA-conditioned T cells.

The pmel-1 T cells that were DCA conditioned showed a marked improvement in tumor clearance and survival. Recurrence upon re-challenge with B16 melanoma was prevented because DCA-conditioned cells induced robust immunologic memory in the tumor. Preliminary results showed similar effects in human peripheral blood mononuclear cells.

On comparing DCA-conditioned T cells and untreated T cells in the same in vivo environment, it was noted that the former had very few phenotypic and functional changes despite significant therapeutic improvement, relative to the latter. DCA-expanded T cells showed a similar level of mitochondrial mass, coinhibitory molecule expression, and total cytokine production post-re-stimulation. Significant quantitative, but not qualitative, benefits of DCA-conditioning were noted among T cells.

After 1 day after infusion, it was noted that DCA-conditioned T cells had a numerical advantage over therapeutic T cells. This numerical advantage also translated to greater long-term persistence. In the absence of any antigen, DCA-conditioned therapeutic T cells were more representative, with a ratio of 8:1.

Concerning the underlying mechanism behind the survival benefit, emerging research suggests that T cells thrive on physiologic carbon sources (PCSs) such as pyruvate, lactate, glutamine, and ketone bodies. Notably, monocarboxylate transporters like MCT1 transport lactate, pyruvate, and ketones. These transporters also export aerobic-glycolysis-derived lactate. It could, therefore, be that DCA-conditioned T cells utilize more PCSs.

It was noted during the expansion that DCA-conditioned T cells were less suitable to ferment glucose into lactate and more to metabolize PCSs. Overall, it seems that by tempering aerobic glycolysis during expansion, therapeutic T cells bear a metabolic profile. This subsequently allows them to re-engraft with greater efficiency.

DCA conferred several key improvements to T cells related to reduced exhaustion gene signature and improved stemness and pre-memory formation. DCA reduced the expression of many terminal exhaustion markers, enhanced the expression of SLAMF6, and slightly enhanced the expression of TCF-1, which are markers of a stem-like profile.

DCA improved the formation of central memory-like T cells, but while studying similar markers for memory formation in human T cells, this effect was not as clear. The amount of cytokine production after re-stimulation with cognate antigen was also enhanced by DCA, contrary to previous findings.

Deeper epigenetic comparisons revealed that histone modifications H3K27Ac and H3K9Ac were elevated in DCA-conditioned T cells. By redirecting pyruvate into the mitochondria, DCA inhibits the production of lactic acid. DCA conditioning was seen to facilitate an epigenetic phenotype because lactate is an epigenetic modifier.

It was hypothesized that DCA may change the epigenetic landscape through carbon flux. This influx does not just concern only the mitochondria but also the nucleus through citrate export into the cytosol. DCA was seen to enhance the quantity of citrate released into the cytosol. The mitochondrial levels of citrate were maintained fairly constant. Importantly, DCA and benzyl-trycarboxylic acid (BTA) conditioning raised respiratory capacity more than DCA alone.

Conclusions

This study identified glucose usage to be the crucial factor for the differential T cells expansion between in vitro and in vivo. The PDHK1 was found to be the vital metabolic node that drives sustained aerobic glycolysis of in vitro-expanded T cells. Therefore, inhibition of PDHK1 during in vitro expansion could significantly enhance the metabolic and functional capacity of ACTs.