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1 Cells glucose glucose glucose 6-phospho glycogen pentose phosphate T 1-phosphate 6-phosphate gluconate CC T CC+PD-1 pathway Isobar: fructose 1,6-diphosphate; glucose 1,6-diphosphate DHAP lactate fructose 6-phosphate fructose 1,6-bisphosphate glyceraldehyde 3-phosphate 1,3-bisphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate PEP pyruvate glycolysis T CC CC+PD-1 CC T CC+PD-1 Supplementary figure 1. PD-1 inhibits glycolytic metabolism during T cell activation. Analysis of key metabolites involved in glycolysis was performed in cells and culture supernatants as described in Materials and Methods. The amounts of the indicated metabolites in unstimulated, T CC and T CC+PD1 cells were plotted in whisker boxes. The glycolytic pathway is shown in parallel.
2 Cells valine BCAT cytosol 3-methyl-2-oxobutyrate mitochondria BCKD isoleucine BCAT 3-methyl-2-oxovalerate BCKD leucine BCAT 4-methyl-2-oxopentanoate BCKD isobutyryl-coa 2-methylbutyryl-CoA isobutyrylcarnitine isovaleryl-coa 3-hydroxyisobutyrate tiglyl-coa 3-methylcrotonyl-CoA gluconeogensis TCA cycle energy fatty acid synthesis Supplementary figure 2. PD-1 inhibits utilization of branched-chain amino acids during T cell activation. The quantities of valine, isoleucine and leucine and the relevant keto-acids generated upon metabolic utilization of the branched-chain amino acids were analyzed in, T CC and T CC+PD-1 cells and their culture supernatants and were plotted in whisker boxes. The integration steps of these keto-acids in metabolic pathways are shown in parallel.
3 Cells docosapentaenoate (n3dpa;22:5n3) fatty acid uptake cytosol mitochondria triacylglycerides membrane phospholipids free fatty acid acyl-coa acyl-carnitine acyl-coa fatty acid synthesis malonyl-coa acetyl-coa.5 - fatty acid acetyl-coa TCA cycle T β-oxidation CC T CC+PD-1 BHBA In excess ketogenesis citrate hydroxybutyrate (BHBA) Supplementary figure 3. PD-1 inhibits lipid biosynthesis and promotes fatty oxidation during T cell activation. The amounts of n3dpa;22:5n3 and 3-hyroxybutyrate (BHBA) were analyzed under the indicated culture conditions and values were plotted in whisker boxes. A schematic outline of fatty acid metabolism is shown in parallel.
4 Palmitate Oxidation (CPMx1 3 /1x1 6 cells) unstim Palmitate Oxidation (CPMx1 3 /1x1 6 cells) a Stimulation Time (hours) c 65 kda - 4 kda - Stimulation Time (hours) T CC T CC +SB T CC +DMSO T CC T CC +PD1 T CC +PD1 +apd Blot Ab: CPT1A b-actin Blot Ab: d b T CC T CC T CC+SB T CC+DMSO Stimulation time (hr) T CC+PD-1 T CC+PD-1 +apd-1 56 kda - 4 kda - pakt(s437) perk1/ kda - b-actin Stimulation time (hr) Supplementary figure 4. (A-B) Vehicle control and the p38 MAPK specific inhibitor SB2358 do not affect CPT1A expression or FAO upon T cell activation. (A) CD4 + human T cells were cultured with tosylatcivated magnetic beads conjugated with acd3/acd28/igg (T CC ) in the presence of either SB2358 (1 um) or vehicle control (DMSO). Cell lysates were prepared at the indicated time points and expression of CPT1A and b-actin was assessed by SDS-PAGE and immunoblot. Results are representative of two experiments. (B) In parallel experiments, fatty acid b-oxidation rate was examined. Values of T CC, T CC +SB and T CC +DMSO cultures were compared to unstimulated () ( P<.5). (C-D) Blockade of PD-1 restores Akt and Erk1/2 activation and results in reduced rate of FAO. CD4 + primary human T cells were either left unstimulated () or were incubated with magnetic beads conjugated with acd3/acd28/pd-l1-ig (T cells costimulated+pd-1; T CC+PD1 ) alone or in the presence of an anti-pd-1 blocking antibody (J15; ebioscience). Activation of Akt and Erk1/2 was examined with phospho-specific antibodies. Fatty acid b-oxidation rate after culture under the indicated conditions was examined. Results are representative of two experiments.
5 ECAR (mph/min) OCR (pmole/min) OCR/ECAR (pmole/mph) Supplementary figure 5. Effects of LY2942 and UO126 on the bioenergetics of activated T cells. CD4 + primary human T cells were cultured with tosylactivated magnetic beads conjugated with anti-cd2/cd28/igg (T CC ) alone or in the presence of LY2942 (LY), UO126 (UO) or their combination. At 72 hours of culture, extracellular acidification rate (ECAR) and oxygen consumption rates (OCR) were assessed. (B) OCR/ECAR ratio was also determined. Values of each experimental conditions were compared to unstimulated () cells ( P<.5, n = 3) and values in T CC +inhibitor cells were compared to T CC (P<.5, n = 3).
6 Unstim 2h 4h 8h h 48h 72h 96h Isotype apd PD1 Supplementary figure 6. Detectable PD-1 expression on the cell surface is not rapidly induced upon T cell activation. CD4 + primary human T cells were cultured with anti-cd3-plus-anti-cd28 mabs and expression of PD-1 at the indicated time points was examined by flow cytometry. Results are representative of three independent experiments.
7 TCC-6 TCC-12 TCC-24 TCC-48 TCC-72 TCC-96 4hr pre- 4hr pre-6 4hr pre-12 4hr pre-24 4hr pre-48 4hr pre-72 4hr pre hr pre- 24 hr pre-6 24 hr pre hr pre hr pre hr pre hr pre-96 ATGL mrna (fold change) TCC-6 TCC-12 TCC-24 TCC-48 TCC-72 TCC-96 4hr pre- 4hr pre-6 4hr pre-12 4hr pre-24 4hr pre-48 4hr pre-72 4hr pre hr pre- 24 hr pre-6 24 hr pre hr pre hr pre hr pre hr pre-96 SNAT2 mrna (fold change) TCC-6 TCC-12 TCC-24 TCC-48 TCC-72 TCC-96 4hr pre- 4hr pre-6 4hr pre-12 4hr pre-24 4hr pre-48 4hr pre-72 4hr pre hr pre- 24 hr pre-6 24 hr pre hr pre hr pre hr pre hr pre-96 SNAT1 mrna (fold change) a T CC Tpr4hr PD-1 Tpr24h PD-1.5 b c Stimulation time (h) Supplementary figure 7. PD-1 suppresses the expression of SNAT1 and SNAT2 and upregulates the expression of ATGL in pre-activated human T cells. CD4 + T cells were cultured with anti-cd3-and-anti-cd28 mabs for 4 hours or for 24 hours and subsequently were collected, rested for three hours and re-cultured with tosyl-activated magnetic beads (1.5 x 1 5 beads / well) conjugated with anti-cd3, anti-cd28 mabs and PD-L1-IgG2a. In the same experiment CD4 + T cells stimulated with tosyl-activated magnetic beads (1.5 x 1 5 beads/well) conjugated with anti-cd3, anti-cd28 mabs and IgG2a were used as positive control.
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