CD28 Superagonistic Activation of T Cells Induces a Tumor Cell-Like Metabolic Program

Abstract

CD28 superagonist (CD28SA), a therapeutic immunomodulatory monoclonal antibody triggered rapid and exaggerated activation of CD4⁺ effector memory T cells (T_EMs) in humans with unwanted serious adverse effects. It is well known that distinct metabolic programs determine the fate and responses of immune cells. In this study, we show that human CD4⁺ T_EMs stimulated with CD28SA adopt a metabolic program similar to those of tumor cells with enhanced glucose utilization, lipid biosynthesis, and proliferation in hypoxic conditions. Identification of metabolic profiles underlying hyperactive T cell activation would provide a platform to test safety of immunostimulatory antibodies.

Keywords: CD28 superagonist; CD4 effector memory T cells; glycolysis; lipogenesis; oxidative phosphorylation.

FIG. 1.

CD28SA-activated CD4⁺ T_EMs display a hyperactive phenotype. (A) Size (FSC) and granularity (SSC) were quantified by flow cytometry. FSC and SSC were measured on purified human CD4⁺ T_EMs stimulated with anti-CD3 or CD28SA for 48 hours. Percentages ± SD indicate T cell blasts. The data are representative of four independent experiments. (B) Human CD4⁺ T_EMs were stimulated with the indicated concentrations of plate-bound antibodies (anti-CD3 or NIB1412) and proliferation was measured 3 days postactivation by ³H-labeled thymidine incorporation. The vertical axis represents mean cpm ± SD from triplicate wells. The data are representative of four independent experiments (**p < 0.01; unpaired, two-tailed Student's t-test). NIB1412–CD28SA. CD28SA, CD28 superagonist; cpm, counts per minute; FSC, forward scatter; SD, standard deviation; SSC, side scatter; T_EMs, effector memory T cells.

FIG. 2.

CD28SA stimulation maximizes OXPHOS potential. (A) OCR (pMoles/min) of preactivated human CD4⁺ T_EMs were measured using the seahorse XF96 extracellular flux analyzer in real time, under basal conditions and in response to sequential addition of Oligomycin (1 μM), 2,4-DNP (160 μM), and antimycin A and rotenone (1 μM). Mean OCR values representing basal respiration and ATP production are presented as bar charts. The bars represent the mean ± SD. The data are representative of three independent experiments (**p < 0.01; unpaired, two-tailed Student's t-test). (B) IF images of mitochondria. IF images show anti-CD3- and CD28SA-stimulated human CD4⁺ T_EMs stained with MitoTracker (red) and Hoechst (blue); far right quadrant represents merged image of the two stains; scale bars represent 10 μm. (C) Mitochondria were stained with MitoTracker Deep Red or (D) MitoSOX Red Mitochondrial Superoxide Indicator, and the staining intensities were quantified by flow cytometry. Data represent the mean ± SD percentage of cells staining positive for mitochondria, oitoROS, with the mean percentage of mitoROS present in activated CD4⁺ T_EMs. Mean ± SD were obtained from three independent experiments (**p < 0.01; unpaired, two-tailed Student's t-test). (E) Human CD4⁺ T_EMs were stimulated with the indicated concentrations of plate-bound antibodies (anti-CD3 or NIB1412) and incubated in normoxic (20% O₂) or hypoxic (5% O₂) conditions. Proliferation was measured 3 days postactivation by ³H-labeled thymidine incorporation. The vertical axis represents mean cpm ± SD from triplicate wells. The data are representative of three independent experiments (**p < 0.01; unpaired two-tailed t-test). NIB1412–CD28SA. IF, immunofluorescent; mitoROS, mitochondrial reactive oxygen species; OCR, oxygen consumption rates; OXPHOS, oxidative phosphorylation.

FIG. 3.

CD28SA stimulation maximizes glucose utilization. (A) ECAR (mpH/min) of preactivated human CD4⁺ T_EMs were measured using the seahorse XF96 extracellular flux analyzer in real time, under basal conditions, and in response to sequential addition of glucose (10 mM), Oligomycin (1 μM), and 2-DG (100 mM). Mean ECAR values representing glycolysis and glycolytic reserve are presented as bar charts. The bars represent the mean ± SD. The data are representative of three independent experiments (**p < 0.01; unpaired two-tailed t-test). (B) Immunofluorescence images show anti-CD3- and CD28SA-stimulated human CD4⁺ T_EMs at 48 hours postactivation stained with anti-GAPDH (green) and DAPI (blue); scale bars represent 10 μm. Far right quadrant represents merged image of the two stains. (C) Human CD4⁺ T_EMs were stimulated with anti-CD3 or CD28SA for 48 hours. Cells were then incubated with 2-NBDG for 30 mins and the amount of 2NBDG uptake (i) was measured by flow cytometry, with the mean percent of 2-NBDG-positive CD4⁺ T_EMs presented in a bar graph. The bars represent the mean ± SD from three different experiments (**p < 0.01; unpaired two-tailed t-test). Cells were also stained for Glut1 expression (ii). The data are representative of three independent experiments. (D) Basal ECAR and OCR of preactivated human CD4⁺ T_EMs were measured using the seahorse XF96 extracellular flux analyzer in real time, with the (i) ECAR/OCR ratio presented as bar charts. The bars represent the mean ± SD. The data are representative of three independent experiments (*p < 0.05, **p < 0.01; two-tailed unpaired t-test). (ii) Cell viability of anti-CD3- and CD28SA-stimulated human CD4⁺ T_EMs cultured for 24 hours in the presence or absence of glucose. Percentage of viable cells was determined by using Trypan Blue. The data are representative of three independent experiments (**p < 0.01; unpaired two-tailed t-test). NIB1412–CD28SA. 2-DG, 2-deoxy-d-glucose; ECAR, extracellular acidification rate.

FIG. 4.

CD28SA activation induces de novo lipogenesis. (A) OCR (pMoles/min) of preactivated human CD4⁺ T_EMs were measured using the seahorse XF96 extracellular flux analyzer in real time, under basal conditions, and in response to sequential addition of Oligomycin (1 μM), 2,4-DNP (160 μM) and antimycin A and rotenone (1 μM). Etomoxir (200 μM) or medium was injected after 2,4-DNP injection. The SRC (quantitative difference between maximal uncontrolled OCR ± etomoxir treatment and initial basal OCR) was calculated and presented as the percentage contribution of FAO to SRC in bar charts. The bars represent mean ± SD. The data are representative of three independent experiments (**p < 0.01; unpaired two-tailed t-test). (B) Immunofluorescence images show (i) anti-CD3- and CD28SA-stimulated CD4⁺ T_EMs at 48 hours postactivation stained with LipidTOX (green) and DAPI (blue); scale bars represent 10 μm. Far bottom quadrant represents merged image of the two stains. (ii) LipidTOX staining was quantified by flow cytometry. Data represent the mean percent of cells ± SD (n = 3) staining positive for neutral lipids. (C-i) Relative levels of acetyl-CoA in anti-CD3- and NIB1412-stimulated T_EMs at 48 hours postactivation; mean ± SD of triplicates (**p < 0.01). (ii) Expression of p-ACC (250 kDa) and p-ACL (125 kDa) by western blot. (iii) Expression of p-ACC with IL-2 condition, in anti-CD3- and NIB1412-stimulated T_EMs at 48 hours postactivation, and (iv) Expression of p-ACC and p-ACL in human malignant melanoma cells (A375), human ovarian tumor cells (A2780), hepatocellular cells (HepG2), human cervical cancer cells (HeLa), human monocytic leukemia cells (THP-1), human T cell lymphoblast-like cells (Jurkat), and unstimulated T_EMs was determined by western blot. Actin (45 kDa) was used as a loading control. (D) Alpha-ketoglutarate-to-citrate ratio in anti-CD3- and CD28SA-stimulated human CD4⁺ T_EMs at 48 hours postactivation, presented as bar charts. The bars represent the mean ± SD of triplicate measurements (*p < 0.05; unpaired two-tailed Student's t-test). NIB1412–CD28SA. ACC, acetyl-CoA carboxylase; ACL, ATP-citrate lyase; FAO, fatty acid oxidation; SRC, spare respiratory capacity.