Tumor-infiltrating T cells are known to lose functionality in the tumor microenvironment and become dysfunctional or terminally exhausted, limiting antitumor immunity and immunotherapy responses. The drivers behind this process remain largely to be determined. Rivadeneira et al. investigated the role of oxidative stress in T cell function. Their results were recently published in Immunity.

Rivadeneira et al. started by assessing the role of oxidative damage on T cell function using a chemo-optogenetic system, which consists of a fluorogen-activating peptide (FAP) and a targeted activating photosensitizer (FAP-TAPS). The FAP peptide binds with high affinity to malachite green (MG) dye, and when MG is iodinated (MG-2I), it produces singlet reactive oxygen species (ROS) upon excitement with 660 nm light. In this system, the oxidative damage can be produced in specific places, and the FAP protein was targeted to the mitochondrial matrix (mitoFAP) for expression in the mitochondria. CD8⁺ T cells were isolated from transgenic mice and stimulated overnight, and were then exposed to the dye and light, resulting in specific ROS creation in the mitochondria. This resulted in mitochondrial dysfunction, with a decrease in mitochondrial membrane potential and loss of basal oxygen consumption rate and spare respiratory capacity.

Rivadeneira et al. then studied the effects of mitochondrial ROS on T cell function and expansion. When mitochondrial ROS were present, T cells had reduced proliferation in the first 3 days of expansion. After 8 days of expansion, ROS had moved beyond the mitochondria and were present throughout the cell. Functionally, T cells with mitochondrial ROS expressed coinhibitory molecules (e.g., PD-1, TIM3), reduced or no TCF7 expression, and increased expression of CD39. The cells also had lower expression of CD27, suggestive of a senescent, terminally differentiated phenotype. When T cells were restimulated, they had a decreased ability to produce IFNγ, TNFα, and IL-2. These data suggest that mitochondrial oxidative stress in T cells drives terminal differentiation and dysfunction.

The researchers then assessed the downstream mechanisms responsible for driving this T cell dysfunction by assessing whether mitochondrial ROS in T cells causes oxidative damage at telomeres. CD8⁺ T cells with or without mitochondrial ROS were isolated and activated in vitro. Seven days after expansion, cells exposed to ROS had increased levels of the DDR proteins γ-H2AX and 53BP1, specifically at telomeres. This suggests DNA damage at telomeres, as these proteins are recruited to sites of DNA damage or dysfunction. Cells exposed to oxidative damage did not show telomere loss or telomere shortening, but there was increased telomere fragility.

The researchers then isolated progenitor exhausted (Tpex) and terminally exhausted (Texh) CD8⁺ T cells from B16 tumors, 10 days after implantation, as well as antigen-experienced T cells from the spleen. No differences between these cells were detected in terms of telomere length, but the tumoral T cells had higher levels of DDR proteins at telomeres than splenic T cells, and the Texh had higher levels of DDR proteins at telomeres than the Tpex cells. These data suggest that tumoral T cells harbor telomeric dysfunction. Using human melanoma and head and neck tumor samples, the researchers also detected higher levels of 53BP1 at telomeres in tumoral CD8⁺ T cells, compared to peripheral blood or healthy donor T cells. However, in contrast to the mouse data, human T cells from tumors showed reduced telomere length compared with peripheral T cells from healthy controls, which might be a consequence of human T cells having shorter telomere lengths than murine T cells.

To further study telomere damage, a model was utilized in which ROS specifically were produced at the telomeres using the telomer-localizing protein TRF1 linked to FAP. Exposure to ROS at telomeres in CD8⁺ T cells resulted in similar effects as exposure to mitochondrial ROS, with reduced T cell proliferation. At day 7 post-activation, T cells with oxidative stress at telomeres had increased expression of PD-1, TIM3, CD39, and the senescence markers p21 and β-galactosidase. After restimulation, the cells exposed to telomere ROS had lower IFNγ, TNFα, and IL-2 production than control cells. Finally, mouse CD105 CAR-T cells were assessed for their cytotoxicity against B16 melanoma cells and had reduced killing capacity after exposure to ROS at the telomeres.

Similar to the mitochondrial oxidative damage, ROS at telomeres also resulted in the localization of the DDR elements 53bp1 and γ-H2AX to the telomeres, indicative of DNA damage. There were no changes in telomere length, but there was increased telomere fragility. The researchers also found pCHK1/ATR telomeric damage responses in the T cells. ATR promotes cell cycle arrest, allowing cells to repair DNA, which requires p53 activation. The T cells had upregulated p53 starting at 1 hour after induction of oxidative damage, which was followed by increases in its transcriptional targets MDM2 and p21. Gene set enrichment analysis showed upregulation of MYC and p53 targets after 24 hours of oxidative damage, as well as upregulation of pathways related to cell cycle control, DNA repair, and telomere maintenance, and downregulation of glycolysis, T cell receptor complexes, and other metabolic processes.

Based on these results, the researchers then asked whether methods to alleviate oxidative stress at telomeres could prevent T cell dysfunction. They developed a fusion protein of the ROS scavenger glutathione peroxidase (GPX1) (that can neutralize superoxide and peroxide) and TRF1 (to localize GPX1 to telomeres). Pmel-1 T cells were transduced with GPX1-TRF1 and expanded to therapeutic quantities, after which they were transferred into B16 tumor-bearing mice. Seven days after transfer, T cells expressing the fusion protein had reduced DDR proteins at telomeres and increased IFNγ, TNFα, and IL-2 production, compared to controls. The cells also had reduced expression of exhaustion markers SOX4 and KLRA9, and were enriched for genes related to mitochondrial function, DNA repair, and cell cycle. The GPX1-TRF1 Pmel-1 T cells showed enhanced tumor control in mice with B16 tumors, resulting in improved survival.

Together, these data suggest that the dysfunctional state in tumoral T cells may be caused by oxidative damage at telomeres. Further, protection of telomeres from this damage may prevent the development of dysfunctional phenotypes and, therefore, may aid in improving the functionality of immunotherapy strategies using engineered T cells.

Write-up by Maartje Wouters, image by Lauren Hitchings

Meet the researcher

This week, first author Dayana Rivadeneira and lead author Greg Delgoffe answered our questions.

What was the most surprising finding of this study for you?
I think one of the biggest surprises was how telomeres in dysfunctional T cells were not shorter, but damaged, and the damage of the telomeres was sufficient to induce a full-blown DNA damage response, and ultimately T cell hypofunctionality. This link has not yet been fully explored in T cells, and it's what Dayana's group, going forward, is going to be focusing on.

What is the outlook?
The shelterin-targeted antioxidant (GPX1-TRF1) is a genetic construct that can essentially be easily translated into clinical vectors for CAR-T cells. We are currently testing other shelterins and other antioxidants to find the most potent one for potential translation, but I'd say the outlook is quite promising for this technology!

If you could go back in time and give your early-career self one piece of advice for navigating a scientific career, what would it be?
GD: If I could go back in time and give the “early career researcher” version of me some advice, it would be to try not to sweat the small stuff. There will be bumps along the road of your scientific life, but the vast majority of the time, that's all they are, bumps along the road that remind you to keep things on track. If you freak about every little hurdle along the way, you won't see the journey for what it is.

Who or what has been a major source of inspiration or motivation for you throughout your career?
DR: My major inspirations are my parents. They worked very hard to give me the opportunity to come to the US to achieve my research career. I recently lost my father to cancer. He is my biggest motivator to keep working towards a better understanding of this terrible disease and designing better therapeutic approaches.