{
  "abstract": "Background Strategies to enhance metabolic pathways can augment T cell expansion, persistence, and effector function, all crucial for an optimal anti-tumor response. However, our lab and others have uncovered intrinsic metabolic insufficiencies in CD8+ tumor-infiltrating lymphocytes (TILs). TILs possess low activity of the glycolytic enzyme, enolase-1 (ENO-1), contributing to diminished glycolysis, oxidative phosphorylation, and cytokine production compared to acute effector T cells (T Eff).1 We expand on this foundational work through the development and characterization of a metabolic activity-based fluorescent probe, termed REPO, generated by linking an ENO-1 binding small molecule2 to a fluorescent moiety.Methods CD8+ T cells from age-matched untreated mouse spleens (T Naïve, N=3-4), vaccinated mouse spleens (Day 5 TEff, N=4-5), or murine melanoma tumors (Day 14-20 TIL, N=10-15) are pulsed with REPO ex vivo and analyzed by flow cytometry. CD8+ TIL are FACS-sorted into REPOhi and REPOlo populations based on gates set by CD8 TNaïve (REPOlo) and TEff (REPOhi) (figure 1A-B). Sorted REPOhi and REPOlo TIL are stained intracellularly for transcription factors, proliferation markers, and cytokines. In vivo Brefeldin A injections enhance cytokine and cytotoxic molecule staining. Sorted REPOhi and REPOlo T cells from tumors or in vitro stimulations are also used in downstream bioenergetic assays (e.g., Seahorse).Results REPO staining by flow cytometry is low in CD8+ naïve T cells and high in vaccine-generated CD8+ T Eff, with CD8+ TIL falling between the biological negative and positive controls (figure 1A), consistent with the relative ENO-1 activity levels and metabolic fluxes from our previous work.1 Higher REPO staining is associated with increased extracellular acidification rate in Seahorse assays (data not shown, ~2-fold, p<0.01). Intranuclear Ki67 protein level is increased in REPOhi TIL compared to REPOlo TIL (figure 2A). REPOhi TIL also have significantly higher in situ IFN-γ and granzyme B protein levels compared to REPOlo TIL (figure 2A). TIL expressing high levels of TOX and inhibitory receptors are present in the REPOhi and REPOlo populations, but only REPOhi TOXhi TIL retain significant cytokine and cytolysis molecule production (figure 2B).Conclusions REPO is a fluorescent small molecule that differentially stains T cells in a manner consistent with their relative levels of metabolic activity. REPO identifies TILs with superior anti-tumor function across exhaustion states. This metabolism-based probe may reflect anti-tumor capabilities and could be integrated into immunophenotyping workflows to evaluate T cell fitness in preclinical and clinical research.Acknowledgements This work was supported primarily by the Melanoma Research Alliance. We thank the University of Virginia Flow Cytometry Core for technical assistance. Cartoon figures made with BioRender.com.References Gemta LF, et al. Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells. Science Immunology 2019;4:eaap9520.Jung DW, et al. Unique small molecule inhibitor of enolase clarifies its role in fundamental biological processes. ACS Chemical Biology 2013;8:1271-1282.Abstract 836 Figure 1REPO strongly stains metabolically active acute effector CD8+ T cells derived from vaccination and weakly stains quiescent naïve T cells, while murine melanoma-derived CD8+ TILs contain both REPOhi and REPOlo populations. (A) Representative plots demonstrating REPO staining intensity for CD8+ Naïve T cells, CD8+ Day 5 Vaccine-Derived Effector T cells, and CD8+ TIL from murine melanomas.(B) REPOhi and REPOlo TILs are sorted by REPO intensity and stained intracellularlyAbstract 836 Figure 2REPOhi TILs possess higher evidence of proliferation and in situ effector function than REPOlo TILs, even within populations expressing high levels of TOX and inhibitory receptors. (A) Representative plots and quantification of Ki67 [top] and IFN-gamma and GzmB [bottom] levels in REPOhi and REPOlo TIL. (B) High REPO staining associates with improved effector function even in TIL populations defined by high TOX and TIM3 expression",
  "authors": [
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Aaron B Streit"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Lelisa F Gemta"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Marissa Gonzales"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Sara E Kaswan"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Jeffery Brulet"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Claire A Conarroe"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Lydia Kitelinger"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Carly van Wagoner"
    },
    {
      "affiliations": [
        "University of Virginia, Waynesboro, VA, USA"
      ],
      "name": "Feifan Xu"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Aeryon Kim"
    },
    {
      "affiliations": [
        "University of Texas at Austin, Austin, TX, USA"
      ],
      "name": "Ku-Lung Hsu"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Timothy NJ Bullock"
    }
  ],
  "title": "836 A novel glycolytic enzyme-based fluorescent probe identifies functionally superior CD8+ tumor-infiltrating lymphocytes (TILs) across states of exhaustion",
  "uid": "3e668b9f-78ff-506f-8ba9-8087d651519b"
}