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. 2022 Sep 20:13:976628.
doi: 10.3389/fimmu.2022.976628. eCollection 2022.

Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage

Elisabetta Cribioli  1   2 , Greta Maria Paola Giordano Attianese  1   2 , Pierpaolo Ginefra  1   2 , Amandine Signorino-Gelo  1   2 , Romain Vuillefroy de Silly  1   2 , Nicola Vannini  1   2 , Christoph Hess  3   4 , Melita Irving  1   2 , George Coukos  1   2
Affiliations

Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage

Elisabetta Cribioli et al. Front Immunol. .

Abstract

Despite the tremendous success of adoptive T-cell therapies (ACT) in fighting certain hematologic malignancies, not all patients respond, a proportion experience relapse, and effective ACT of most solid tumors remains elusive. In order to improve responses to ACT suppressive barriers in the solid tumor microenvironment (TME) including insufficient nutrient availability must be overcome. Here we explored how enforced expression of the high-affinity glucose transporter GLUT3 impacted tumor-directed T cells. Overexpression of GLUT3 in primary murine CD8+ T cells enhanced glucose uptake and increased glycogen and fatty acid storage, and was associated with increased mitochondrial fitness, reduced ROS levels, higher abundance of the anti-apoptotic protein Mcl-1, and better resistance to stress. Importantly, GLUT3-OT1 T cells conferred superior control of B16-OVA melanoma tumors and, in this same model, significantly improved survival. Moreover, a proportion of treated mice were cured and protected from re-challenge, indicative of long-term T cell persistence and memory formation. Enforcing expression of GLUT3 is thus a promising strategy to improve metabolic fitness and sustaining CD8+ T cell effector function in the context of ACT.

Keywords: T cell engineering; adoptive cell therapy; glucose; immunotherapy; metabolism; tumor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Enforcing expression of GLUT3 enhances glucose uptake by TEM cells. (A) Schematic of pMSGV retroviral vectors encoding Thy1.1 (T cell engineering control termed MOCK), and GLUT3 comprising a Flag tag. (B) Schematic of OT1 T cell isolation, activation and expansion in high-dose IL-2. (C) Evaluation of transduction efficiency for GLUT3 expression by anti-Flag antibody staining and flow cytometric analysis. (D) Viability of transduced T cells as measured by staining with a viability dye and flow cytometric analysis (days 9-12). (E) Percentage of CD44+ CD62L+ and CD44+ CD62L- T cells in culture. Left: summary of all donors tested. Right: representative flow cytometry dot plot. (F) Left: glucose uptake by T cells evaluated with the fluorescent glucose analog 2-NBDG with or without the addition of the glucose competitor 2-DG. Right: representative histograms showing 2-NBDG uptake. (G) Glucose uptake by T cells measured in a luminescence-based assay. Shown is average ± standard deviation (SD) of different cultures. For data in which fold-change is shown, values of GLUT3 expressing T cells of each culture are normalized with the values of the corresponding MOCK-T cells. Shown is average ± standard deviation (SD) of different cultures. Statistical analysis by paired, two-tailed t test (F: MOCK vs GLUT3 p=0.001, MOCK vs MOCK+2DG p= 0.0029, GLUT3 vs a GLUT3 +2DG p=0.0138). **p< 0.01; *p < 0.05.
Figure 2
Figure 2
Enforcing expression of GLUT3 is associated with elevated energy storage by TEM cells. (A) Glucose content in T cell lysate measured with a colorimetric assay. (B) Glycogen content in T cells as evaluated with a colorimetric assay. (C) Left: summary of periodic acid shiff (PAS)-based evaluation of glycogen in MOCK- versus GLUT3-T cells. Right: representative 100X images of PAS staining of MOCK- versus GLUT3-T cells. Red arrow indicates an example of a PAS positive cell (magenta), blue arrow indicates example of a negative cell (purple, hematoxylin positive). (D) Representative transmission electron microscopy pictures (magnification 3800X) of MOCK- versus GLUT3-T cells. White arrows point to glycogen deposits. (E) Intracellular staining for pGSK3β (at Ser9). (F) Correlation between glycogen content and percentage of transduction. (G) Left : mean fluorescence intensity (MFI) for T cells stained with Bodipy 493/503 dye. Right: representative histogram of Bodipy 493/503 dye staining. (H) Size of MOCK- versus GLUT3-T cells (μm). For data in which fold-change is shown, values of GLUT3 expressing T cells of each culture are normalized with the values of the corresponding MOCK-T cells. Shown is average ± SD of different cultures. Statistical analysis by paired, two-tailed t test (A–C, E, G, H), Pearson correlation (r=correlation coefficient) (F). **p< 0.01; *p < 0.05.
Figure 3
Figure 3
Enforcing expression of GLUT3 is associated with higher mitochondrial polarization and improved in vitro function of TEM cells. (A) Staining of T cells with TMRM and Mitotracker green (MG). Left: TMRMhighMGhigh values for GLUT3-T cells are normalized with the values for MOCK-T cells from the same donor. Right: representative dot plot of TMRM and MG stained T cells. Upper gating represents the TMRMhighMGhigh population. (B) Percentage of TMRMhighMGhigh T cells following overnight incubation with 2-DG (C) Left: quantification of intracellular reactive oxygen species (ROS) with CellROX Green reagent. Right: representative plot of CellROX staining. (D) Left: evaluation of Mcl-1 levels by intracellular staining. Right: representative plot of anti-Mcl-1 antibody-stained cells. (E) Left and middle: Analysis of IFNγ and TNF production by T cells upon overnight stimulation with SIINFEKL peptide in media deprived of glucose (Glc), media with low Glc (Glc= 0.444 mM), or complete media (Glc= 11.1 mM). Right: representative dot plot of anti-IFNγ and anti-TNF antibody staining upon stimulation with SIINFEKL peptide in different media. Shown is one representative experiment out of three. (F) Left: mean cell division of T cells stimulated for 5 days with irradiated B16-OVA tumor cells in media deprived of Glc, media with low Glc concentration, or complete media. Right: representative histograms of cell trace violet (CTV) dilution proliferation experiment. Shown is one representative experiment out of three. Shown is average ± SD of different cultures. Statistical analysis by paired, two-tailed t test (A, C, D), two-way analysis of variance (ANOVA) with correction for multiple comparisons by post hoc Tukey’s test (B: MOCK vs MOCK+2-DG p=0.0131; MOCK vs GLUT3 p=0.0089; GLUT3 vs GLUT3+2-DG p= 0.0049; MOCK+2-DG vs GLUT3+2-DG p=0.0235), two-way ANOVA with correction for multiple comparisons by post hoc Sidak’s test (E, F). **p< 0.01; *p < 0.05; ns, non -significant.
Figure 4
Figure 4
Enforcing expression of GLUT3 enables higher glucose uptake, survival, and resistance to exhaustion of TCM cells. (A) Schematic for the generation of GLUT3-TCM cells. (B) Evaluation of transduction efficiency for GLUT3 expression by anti-Flag antibody staining and flow cytometric analysis. (C) Viability of transduced T cells as measured by staining with a viability dye and flow cytometric analysis (days 9-12). (D) Percentage of CD44+ CD62L+ and CD44+ CD62L- T cells in culture. Left: summary of all donors tested. Right: Representative flow cytometry dot plot. (E) Seahorse analysis of the basal extracellular acidification rate (ECAR) of MOCK- and GLUT3-T cells with a metabolic perturbation assay. (F) Top Left: Percentage necrosis (AnnexinV-/7-AAD+) of MOCK- versus GLUT3-T cells over time in media with or without glucose (Glc).Bottom Left: evaluation of live (Annexin V-/7-AAD-), early apoptotic (Annexin V+/7-AAD-), late apoptotic (Annexin V+/7-AAD+), and necrotic (Annexin V-/7-AAD+) MOCK- and GLUT3-T cells after 72h in culture in the presence or absence of Glc. Right: representative dot plot of AnnexinV/7-AAD staining at 72h (AnnV=Annexin V). (G) Expression levels of PD1, LAG3, TIM3, TCF-1 and TOX upon repeated stimulation with SIINFEKL peptide for 5 days. Top: mean fluorescence intensity (MFI) levels are shown. MFI levels of GLUT3-T cells from each culture are normalized to corresponding MOCK-T cells. Bottom: representative histograms of PD1, LAG3, TIM3 TCF1 and TOX expression. Shown is average ± SD of different cultures. Statistical analysis by paired, two-tailed t test (E, G), two-way analysis of variance (ANOVA) with correction for multiple comparisons by post hoc Tukey’s test (F Top Left: Glc+ 48h p=0.0005, Glc- 48h p<0.0001, Glc+ 72h p<0.0001, Glc- 72h p<0.0001), two-way ANOVA with correction for multiple comparisons by post hoc Sidak’s test (F Bottom Left: Glc +, Necrosis MOCK vs GLUT3 p=0.0016; Glc +, Early Apoptosis MOCK vs GLUT3 p=0.0009; Glc -, Necrosis MOCK vs GLUT3 p=0.0456). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, non-significant.
Figure 5
Figure 5
Enforcing expression of GLUT3 by TCM cells enhances tumor control and survival of mice treated by ACT. (A) Schematic of adoptive cell transfer (ACT) study. (B) Tumor growth over time in individual mice following ACT. Left: ACT of MOCK-T cells. Right: ACT of GLUT3-T cells. Dotted grey line marks 30 days post tumor cells injection. (C) Kaplan-Meier analysis of mice survival up to 110 days after tumor cell injection. (D) Growth and control of tumors in the right flanks of 4 mice surviving in 2 independent ACT studies. (E) Schematic of tumor cell rechallenge experiment in 4 mice survivors. Mouse survivors 1 and 2 (orange dot and yellow square) were newly injected at day 110 post their initial tumor cell injection (previously on their right flanks), and mouse survivors 3 and 4 (pink and purple triangles) at 170 days. (F) Tumor growth curves for survivors and control mice. (G) T cell persistence in the lymph nodes and spleens of survivors at 50 days post rechallange as assessed by anti-CD45.1 antibody staining and flow cytometric analysis. (H) Representative anti-Flag tag antibody staining of T cells extracted from the lymph nodes of survivors. Statistical analysis by Log-rank (Mentel-Cox) (C). *p < 0.05.
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References

    1. Lanitis E, Dangaj D, Irving M, Coukos G. Mechanisms regulating T-cell infiltration and activity in solid tumors. Ann Oncol (2017) 28:xii18–32. - PubMed
    1. Pearce EL, Poffenberger MC, Chang CH, Jones RG. Fueling immunity: Insights into metabolism and lymphocyte function. Science (2013) 342:1242454. - PMC - PubMed
    1. Salmond RJ. mTOR regulation of glycolytic metabolism in T cells. Front Cell Dev Biol (2018) 6:122. - PMC - PubMed
    1. Dimeloe S, Burgener AV, Grählert J, Hess C. T-Cell metabolism governing activation, proliferation and differentiation; a modular view. Immunology (2017) 150:35–44. - PMC - PubMed
    1. Zhang L, Romero P. Metabolic control of CD8+ T cell fate decisions and antitumor immunity. Trends Mol Med (2018) 24:30–48. - PubMed

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