Autophagy metabolically suppresses CD8⁺ T cell antitumor immunity

ABSTRACT

Macroautophagy/autophagy is a critical regulator of adaptive T cell immunity and homeostasis. However, the role of T cell autophagy in regulating antitumor immune responses is less clear. In a recent study, we showed that deletion of the essential autophagy genes Atg5, Atg14, or Atg16l1 in host tissues dramatically impairs growth of autophagy-competent syngeneic tumors. We further demonstrated that CD8⁺ T cells lacking Atg5 acquire an effector memory phenotype and produce more IFNG/IFN-γ (interferon gamma) and TNF/TNF-α (tumor necrosis factor). These phenotypic changes are accompanied by enhanced glucose metabolism that results in alterations in histone methylation, and upregulation of glycolytic and immune response genes. In accordance with this, we observed control of tumor growth in autophagy-competent mice after adoptive transfer with a sub-therapeutic dose of atg5^-/- T cells. Collectively, we discovered a unique, cell-autonomous role for T cell autophagy in the metabolic control of antitumor immunity.

KEYWORDS:

• Antitumor immunity
• autophagy
• CD8⁺ T cells
• glycolysis
• lactate
• methylation
• SAM

Macroautophagy (autophagy) is a key salvage pathway in which cytoplasmic components are catabolically degraded in lysosomes for recycling. Autophagy is upregulated in response to nutrient deprivation or hypoxia, and promotes cell survival under stressful conditions. In cancer cells, autophagy has both pro- and anti-tumorigenic effects. However, the role of T cell autophagy in regulating antitumor immune responses is less clear. A recent study by our group uncovered an unexpected role for autophagy as a negative regulator of CD8⁺ T cell effector function.

Initially, we sought to examine the role of autophagy in tumor growth. We generated a tamoxifen-inducible atg5 knockout mouse to delete Atg5 in all host tissues. Control (Atg5^±) and knockout (atg5^-/-) mice were implanted with autophagy-competent e0771 breast or Tramp-C2 prostate cancer cells, and although rates of tumor initiation are similar, overall tumor growth is significantly impaired in atg5^-/- mice. We saw similar tumor clearance in mice with inducible Atg16l1 deletion, suggesting that loss of canonical autophagy promotes tumor rejection.

To further define the effects of autophagy on the immune compartment, we generated bone marrow chimeric (BMC) mice by transferring either control (Atg5^±) or knockout (atg5^-/-) bone marrow into wild-type (WT) host mice. Similar to the whole-body knockout, tumor growth is significantly reduced in atg5^-/- BMC mice compared to control Atg5^± BMC mice. However, when atg5^-/- mice receive WT bone marrow, the observed reduction in tumor growth is completely abrogated. These experiments were corroborated using Atg14 BMC mice, collectively demonstrating that autophagy in this system is essential in mediating antitumor immune responses.

In accordance with previous studies, we saw a decrease in the total number of CD4⁺ and CD8⁺ T cells in all compartments (blood, spleen, and tumor) following inactivation of autophagy. To understand how reduced T cell number can correlate with the enhanced antitumor responses observed in vivo, we profiled tumor infiltrating lymphocytes (TIL) from atg5^-/- mice. More than 80% of CD8⁺ TIL acquire an effector memory (SELL/CD62L^lo CD44^hi) phenotype, while no change in the frequency of effector memory CD4⁺ TIL is observed. As a result of the shift to an effector memory phenotype, atg5^-/- CD8⁺ T cells from tumor-bearing mice produce more IFNG and TNF. Elevated production of IFNG is also observed in CD8⁺ TIL from atg14^-/- BMC mice, indicating that loss of autophagy enhances CD8⁺ T cell effector function.

To determine whether the increased antitumor activity is due to cell autonomous loss of autophagy, we implanted SERPIN/ovalbumin-expressing EL4 tumors in WT mice, and observed tumor control after only one sub-therapeutic dose of atg5^-/- OT1 (ovalbumin-specific) T cells. In contrast, tumors progressively increase in size in mice that receive Atg5^± OT1 T cells. Consistent with this observed increase in antitumor response, atg5^-/- OT1 T cells isolated from tumor-bearing mice produce more IFNG.

Mechanistically, atg5^-/- CD8⁺ T cells adopt a unique metabolic phenotype characterized by enhanced glucose metabolism. atg5^-/- CD8⁺ T cells isolated from tumor-bearing mice have a significant increase in glycolysis as shown by the reduction in the ratio of oxygen consumption rate to extracellular acidification rate. Correspondingly, both atg5^-/- and atg14^-/- T cells take up more of the fluorescent glucose analog 2-NBDG. After culturing atg5^-/- CD8⁺ T cells under conditions of glucose starvation, a 3-fold reduction in IFNG production is observed, indicating that glucose metabolism is required to maintain enhanced cytokine production in the absence of Atg5.

These observations suggest that autophagy deletion leading to enhanced antitumor immune responses by CD8⁺ T cells can be due to metabolic-mediated changes in the transcriptional regulation of immune regulatory pathways. Indeed, metabolomic profiling revealed that atg5^-/- CD8⁺ T cells exhibit significant alterations in metabolite levels. Among them, methionine, an essential metabolite for the production of the ubiquitous methyl donor S-adenosylmethionine, displays higher abundance in atg5^-/- T cells compared to Atg5^± T cells. This observation prompted us to ask whether epigenetic modifications could explain the dramatic changes in atg5^-/- T cell effector function. Using chromatin immunoprecipitation sequencing, a total of 466 promoters were found to be uniquely enriched for H3K4me3 (an activating mark) in atg5^-/- TIL as compared to Atg5^± TIL. Further gene ontology and pathway analysis highlighted a remarkable collection of genes involved in T cell activation and immune response as those most significantly enriched for H3K4me3 in atg5^-/- T cells. In contrast, few localized differences were observed in signal density of H3K27me3, an inhibitory mark.

In conclusion, we propose that inactivation of autophagy prevents metabolic downregulation of H3K4me3 levels, thus enabling the maintenance of H3K4me3 density at promoters of immune response genes. Our unexpected results support modulation of CD8⁺ T cell autophagy as a novel strategy to improve T cell-based immunotherapies.

Acknowledgments

The authors were supported by the Canadian Breast Cancer Foundation and the BC Cancer Foundation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Canadian Breast Cancer Foundation [n/a]; BC Cancer Foundation [n/a].