F-actors in action: being CD8 means to switch energy needs for every occasion

Killer immune cells destroy cancer cells and cells infected by virus. These CD8⁺ T cells are activated after detection of viral infection or growth of “non-self” tumor cells. However, in chronic viral infection and cancer, the killer cells often lapse into “exhausted” CD8⁺ T cells that no longer can stem disease. This exhaustion is a major barrier in the new immunotherapies for cancer, including immune checkpoint blockers and CAR T cell therapy. In a detailed study of exhausted T cell subsets, University of Alabama at Birmingham researchers show that a transcriptional repressor called Gfi1 is a key regulator of the subset formation of exhausted CD8⁺ T cells and may offer a key to reducing exhaustion.

Exhausted CD8⁺ T cells are a complex population of subsets composed of progenitor cells and “effector-like” or “terminally exhausted” cells. Effector-like cells still retain some killer ability. The UAB researchers used mice infected with a chronic virus to describe four subsets in the population, including a previously under-described Ly108⁺CX₃CR1⁺ subset that expresses low levels of Gfi1, while other established subsets have high expression. Notable key features of the Ly108⁺CX₃CR1⁺ subset include: First, the Ly108⁺CX₃CR1⁺ subset has a distinct chromatin profile from the other sets, meaning a changed accessibility to certain genes on the chromosome. Second, that subset is transitory and develops to terminally exhausted cells and effector-like cells, which retain some tumor killing ability. Third, this process depends on Gfi1.

To demonstrate a role for Gfi1 in immune checkpoint blockade therapy, the UAB team tested anti-CTLA-4 therapy in a mouse bladder cancer model, comparing mice that had T cells with either wild-type Gfi1 or Gfi1 knockout. They found that the anti-CTLA-4 therapy significantly inhibited tumor growth in wild-type but not Gfi1 knockout mice. Similarly, anti-CTLA-4 therapy promoted infiltration and/or expansion of CD4⁺ and CD8⁺ tumor-infiltrating lymphocytes in wild-type but not Gfi1 knockout mice. Considering Gfi1 downregulation is associated with the active differentiation of CD8⁺ T cell progenitors, scientists argued that transient and intermittent inhibition of Gfi1 with an histone demethylase (scientists deem it might be LSD1) may facilitate the differentiation of progenitors to Ly108⁺CX₃CR1⁺ cells and then to effector-like cells, thereby improving the control of tumors.

These observations were largely corroborated in a second mouse model of colorectal adenosarcoma MC38. Along with a recent report by others of promising outcomes in small cell lung cancer from combining a histone demethylase inhibitor with the anti-PD-1 immune checkpoint blocker, further testing of this combination approach should be conducted in melanoma, bladder cancer and colorectal adenocarcinoma, especially those resistant to immune checkpoint blockers.  In another set of experiments, ablation of Gfi1 resulted in downregulation of the transcription factor EOMES and apoptosis suppressor Bcl-2 in memory CD8⁺s. Ectopic expression of EOMES rescued the expression of Bcl-2, but the persistence of memory CD8⁺ T cells was only partially rescued.

These findings highlight the critical role of GFI1 in the long-term maintenance of memory CD8⁺ T cells in persistent infections by sustaining their proliferative potential. But to understand the genetic workout one must firstly understand or elucidate upstream signalings derived also from energy sources availability. For cancer- and infection-fighting T cells, glucose offers far more than a simple sugar rush. T cells activated in vitro predominantly metabolize glucose via aerobic glycolysis, leading to increased lactate production, a process known as the Warburg effect. However, in addition to glycolysis, activated T cells also increase oxidative phosphorylation (OXPHOS), which is fueled, in part, by glucose-derived pyruvate.

Interestingly, compared with in vitro-activated CD8⁺ T cells, CD8⁺ T cells activated in vivo produce less lactate and have increased OXPHOS rates. Under physiologic conditions, however, CD8⁺ T cells preferentially oxidize non-glucose carbon sources to fuel the tricarboxylic acid (TCA) cycle and OXPHOS. In the presence of these alternate carbon sources, glucose is prioritized to fuel biosynthetic pathways associated with cell growth and proliferation, such as nucleotide and lipid biosynthesis, while still contributing to cellular bioenergetics via glycolysis-dependent ATP and NADH production. Together, these studies suggest that CD8 T cells have distinct requirements for glucose in vivo; however, the metabolic fates of glucose critical for Teff cell function under physiologic conditions remain poorly defined.

A new discovery by Van Andel Institute scientists reveals that glucose, an essential cellular fuel that powers immune cells, also aids in T cells’ internal communication and boosts their cancer-fighting properties. The findings may help optimize T cells’ ability to combat cancer and other diseases and reveal that T cells allocate significant portions of glucose to build large molecules called glyco-sphingolipids (GSLs). These sugar-fat compounds are essential for T cell growth and making proteins that T cells use to combat cancer. GSLs help form fat-rich structures on T cell surfaces called lipid rafts, which bring together cell signaling proteins that instruct the T cell to kill cancer cells. Without GSLs, these signals are weaker, making T cells less effective at destroying tumors.

To evaluate whether impaired lipid rafts translated to defects in TCR signaling, the research team used enzyme-deficient (UGCG) CD8⁺ T cells. An early event in TCR signaling is the phosphorylation and activation of ZAP70 kinase. Activated ZAP70 subsequently phosphorylates LAT, a lipid raft-associated transmembrane protein that recruits other proteins such as Grb2 adaptor and phospholipase C gamma 1 (PLCγ1). Recruitment of PLCγ1 to LAT in lipid rafts is required for PLCγ1 phosphorylation and Grb2 recruitment leads to activation of c-Jun N-terminal kinases (JNKs). Control and UGCG-deficient CD8 OT-I T cells displayed similar kinetics of ZAP70 phosphorylation in response to OVA peptide stimulation. By contrast, PLCγ1 phosphorylation was reduced and c-Jun phosphorylation delayed in UGCG-deficient OT-I cells following stimulation.

As an orthogonal approach, the team assessed TCR signaling in CD8⁺ OT-I T cells in which lipid rafts were disrupted using eliglustat. Like Ugcg deletion, eliglustat dampened OVA-induced PLCγ1 and c-Jun phosphorylation in a concentration-dependent manner. Taken together, data demonstrate that impairing lipid rafts by targeting GSL biosynthesis hinders distal TCR signaling. Depleting GSLs reduced phosphorylation of c-Jun, a component of the AP-1 transcription factor complex, following TCR stimulation. In coordination with other transcription factors activated by TCR downstream signaling (e.g., NF-ATc), AP-1 regulates the expression of genes involved in cell proliferation, migration and effector function.

Both T cells and cancer cells leverage different nutrients to support varying aspects of their function, even though glucose remains the fastest and the preferred one. Curiously, either immune and cancer cells utilize free fatty acids as energy sources when glucose is low. For example, some prostate and breast cancer cells have a parallel dependence on free fatty acids to enhance their growth. The more scientists know about these different fuel sources, the better we can support T cells’ innate cancer-fighting abilities while also developing ways to possibly make cancer cells more vulnerable to immune attack. For example, an earlier work of the same team with Dr. J Longo showed that CD8 Teff cells use metabolites for specific pathways during specific phases of activation.

Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the TCA cycle to support ATP and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1) and change fuel preference over the course of infection, switching from glutamine to acetate-dependent TCA cycle metabolism late in infection. This provides scientists with insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

• Edited by Dr. Gianfrancesco Cormaci, PhD, specialista in Clinical Biochemistry.

Scientific references

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