SUPPLEMENTARY WEB MATERIAL

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1 SUPPLEMENTARY WEB MATERIAL Rapid effector memory CD8+ T cell function requires an immediate-early glycolytic switch Patrick M. Gubser 1, Glenn R. Bantug 1, Leyla Razik, Marco Fischer, Sarah Dimeloe, Gideon Hoenger, Bojana Durovic, Annaïse Jauch & Christoph Hess 1 These authors contributed equally to this work 1

2 SUPPLEMENTARY FIGURES Supplementary Figure 1. Sorting strategy for CD8 + T cell populations. a, Representative dot plot of fresh isolated CD8 + T cells stained for CD45RA and CCR7. Four CD8 + T cell subpopulations are identified as follows: naïve (CCR7 + CD45RA + ), central memory (CM, CCR7 + CD45RA neg ), effector memory (EM, CCR7 neg CD45RA neg ) and CD45RA re-expressing EM (EMRA, CCR7 neg CD45RA + ). Values represent frequency of total gated events. b-c, Representative dot plots of naïve (b) and EM (c) CD8 + T cell subsets sorted by FACS (purity always greater than 95%). d, Representative dot plot of fresh isolated CD8 + T cells stained for CD45RA and CD62L (different donor than in (a)). As above, four CD8 + T cell subpopulations are identified as follows: naïve (CD62L + CD45RA + ), CM (CD62L + CD45RA neg ), EM (CD62L neg CD45RA neg ) and EMRA (CD62L neg CD45RA + ). Values represent frequency of total gated events. e-f, Representative dot plots of CM (e) and EM (f) CD8 + T cell subsets sorted by FACS (purity always greater than 95%). Values represent frequency of total gated events. 2

3 Supplementary Figure 2. Schematic diagram of OCR and ECAR profiles as generated by the Seahorse extracellular flux analyzer. a, Schematic OCR time course under basal conditions and following perturbation of mitochondrial respiration with oligomycin, DNP and rotenone. Using this perturbation profiling technique, four OCR rates are directly measured: the non-corrected basal OCR [OCR(basal-nc)], the rate following inhibition of ATP synthase [OCR(oligomycin)], the peak rate following mitochondrial uncoupling [OCR(peak-DNP)], and the rate following inhibition of mitochondrial respiration [OCR(rotenone)]. The following respiratory parameters (indicated by red double-ended arrows in the diagram) are calculated using the formulas below: (1) basal respiration = [OCR(basal-nc)] [OCR(rotenone)] (2) ATP coupled respiration = [OCR(basal-nc)] [OCR(oligomycin)] (3) leak respiration = [OCR(oligomycin)] [OCR(rotenone)] (4) maximal respiratory capacity = [OCR(peak-DNP)] [OCR(rotenone)] (5) spare respiratory capacity = [OCR(peak-DNP)] [OCR(basal-nc)] (6) non-mitochondrial respiration = OCR(rotenone) b, Schematic ECAR time course under basal conditions and following inhibition of mitochondrial respiration. Basal ECAR is the initial rate measured by the extracellular flux analyzer. The maximal ECAR is the rate following addition of rotenone. Red double-ended arrows in the diagram indicate the respective glycolytic parameters. 3

4 Supplementary Figure 3. Calculated respiratory parameters of naïve and EM CD8 + T cells. a-c, Key respiratory parameters were calculated following perturbation of mitochondrial function with oligomycin, DNP, and rotenone. Graphs show (a) ATPcoupled respiration, (b) leak respiration and (c) non-mitochondrial respiration of naïve and EM CD8 + T cells (n = 6 separate donors, paired two-tailed Student's t-test). Supplementary Figure 4. CD28 expression on naïve and EM CD8 + T cells. a, Representative histograms of naïve (red) and EM (blue) CD8 + T cells stained for cellsurface expression of CD28. b, Graph of CD28 mean fluorescence intensities from both subsets (n = 9). c, Bar graph showing frequencies of CD28 positive cells in both subpopulations (n = 7, ± s.d.). 4

5 Supplementary Figure 5. Incubation of naïve and EM CD8 + T cells with isotype control mab and anti-cd28 mab did not induce glycolytic switch. ECAR of naïve ( ) and EM ( ) CD8 + T cells following in-seahorse stimulation with isotype control mab (2 µg/ml) and anti-cd28 (20 µg/ml) mab (means ± s.e.m.). Representative of 3 independent experiments (separate donors). Supplementary Figure 6. Long-term treatment with rapamycin diminishes activation induced, glycolytic switch in CD8 + T cells. a-b, Representative mean ECAR of activated CD8 + T cells. (a) Naïve and (b) EM CD8 + T cells were activated with anti- CD3 mab (1 µg/ml) and anti-cd28 mab (10 µg/ml), or left unstimulated for 3 days and then processed for Seahorse. After measuring basal ECAR, cells were in-seahorse restimulated with anti-cd3 and anti-cd28 mab (as above), followed by treatment with 25 mm 2-DG at approximately 300 minutes post-mab injection. Non-activated (, ), anti-cd3 and anti-cd28 mab activated (, ), and anti-cd3 and anti-cd28 mab plus 20 ng/ml rapamycin (, ). Open and closed symbols represent naïve and EM CD8 + T cells, respectively. Representative of 2 experiments (separate donors). 5

6 Supplementary Figure 7. Impact of Akt-inhibition on cytoplasmic GAPDH expression in non-activated and activated EM CD8 + T cells. Graphic summary of cytoplasmic GAPDH fluorescence intensity in CD8 + T cells under non-activating conditions incubated in presence and absence of 10 µm Akti 1-2, and following stimulation with anti-cd3 (2 µg/ml) and anti-cd28 (20 µg/ml) mab for 2 h in presence and absence of 10 µm Akti 1-2 (n = 3 separate donors, from all donors a total of n = 35 [non-activated], n = 57 [non-activated; Akti 1-2 treated], n = 62 [activated], n = 77 [activated; Akti 1-2 treated] EM CD8 + T cells were analyzed, error bars = s.e.m., Man- Whitney U-test). *P<0.001 Supplementary Figure 8. Relative contribution of TCR- and CD28-signaling to immediate-early IFN-γ production of EM CD8 + T cells. IFN-γ was quantified in the supernatant of EM CD8 + T cells incubated for 12 h under non-activating conditions, or with isotype for anti-cd3 (0.2 µg/ml) plus anti-cd28 mab (20 µg/ml); anti-cd3 mab alone (0.2 µg/ml); or anti-cd3 plus anti-cd28 mab (0.2 and 20 µg/ml) (n = 3 separate donors, error bars = s.e.m., paired two-tailed Student's t-test). *P<0.05 6