Figure S1. CD28 is not required for TCR-mediated rapid glycolysis in naïve nor previously activated CD8+ T cells. (Supports Figure 2) A, (Left) ECAR trace of ...
Cell Reports, Volume 22
Supplemental Information
Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions Ashley V. Menk, Nicole E. Scharping, Rebecca S. Moreci, Xue Zeng, Cliff Guy, Sonia Salvatore, Heekyong Bae, Jianxin Xie, Howard A. Young, Stacy Gelhaus Wendell, and Greg M. Delgoffe
Figure S1.
ECAR (mpH/min)
A
ECAR (mpH/min) [ECARmax- ECARbasal]
ECAR (mpH/min)
ECAR (mpH/min)
B
Figure S1. CD28 is not required for TCR-mediated rapid glycolysis in naïve nor previously activated CD8+ T cells. (Supports Figure 2) A, (Left) ECAR trace of naïve, CD8+ T cells stimulated with streptavidin or streptavidin crosslinked- αCD3 at 3 μg/ml in the presence or absence of 2 μg/ml αCD28, (right) tabulated results from multiple experiments. B, (Right) tabulated ECAR of naïve, CD8+ T cells stimulated with indicated amounts of streptavidin-crosslinked αCD3 in the presence or absence of αCD28, (left, top) trace ECAR of optimal TCR activation, (left, bottom) trace ECAR of suboptimal activation. Results represent the mean of five (A) or three (B) independent experiments. ** p < 0.01 ns, not significant by unpaired t-test. Error bars represent SEM.
pLDH (Y10) signal [normalized to Actin]
A
αCD3 NS 1 5 10 P
P LDH (Y10) PKM2 (Y105) Actin
B
pPKM2 (Y105) signal [normalized to Actin]
Figure S2.
αCD3 NS 1 IP:
P
IP:
P
C
5 10
Tyrosine IB: Enolase 10% input IB: Enolase
IP:
Tyrosine IB: PGAM1 10% input IB: PGAM1 IP: P Tyrosine IB: Hexokinase II 10% input IB: Hexokinase II
αCD3 NS 1 Tyrosine IB: PDHK1 10% input IB: PDHK1
D P
5 10
+DCA
** *
αCD3
PDHK1 (Y243) P PDH (S293) total PDHK1
0 1 5 10 1
2.0
5 10
1.5 1.0 0.5 0.0
NS 1 5 10 1 5 10 CD3 DCA
Resting, freshly isolated T cells (normal media)
%m+3 13C-lactate [in media]
G
H 100 80
Extract for analysis
Pulse 1:1
30 min
30 min
F
13C-glucose
Anti-CD3 +/- DCA
*
60 min
I
%m+3 13C-pyruvate
E
J
p=0.053
60 40 20 0
NS
CD3 CD3 +DCA
Figure S2. TCR signaling does not induce phosphorylation of glycolytic enzymes but rather reroutes glucose processing. (Supports Figure 3) A, (Left) representative immunoblot (IB) of indicated phospho or total proteins in lysates from previously activated, CD8+ T cells stimulated for the indicated periods with streptavidin-crosslinked αCD3 at 3 μg/ml, (right) tabulated densitometry scanning. B, IB of immunoprecipitations of phospho-tyrosine in lysates from PA-R CD8+ T cells stimulated as in A. C IB of immunoprecipitation of phospho-tyrosine in lysates from PA-R CD8+ T cells stimulated as in A. D, (Top) representative IB of indicated phospho or total proteins in lysates from PA-R CD8+ T cells stimulated as in A for the indicated periods in the presence or absence of 20 mM DCA, (bottom) tabulated densitometry scanning. E, Flow chart for 13C-glucose experiment. Freshly isolated CD8+ T cells were rested 30 min in Seahorse media containing glucose, stimulated with 3 ug/mL anti-CD3 for 30 minutes, and then pulsed with an equimolar concentration (10 mM) of 13C-uniformly labeled glucose. Metabolites were extracted 1 h after pulse. F, 13C labeled pyruvate from experiment described in E. G, 13C labeled lactate from experiment described in E. H, 13C labeled malate from experiment described in E. I, 13C labeled citrate from experiment described in E. J, Unlabeled (m+0) malate and citrate levels from E. Results represent three independent experiments. NS, not significant by unpaired t-test.
Figure S3. A
B
C 30
*
*
ECAR (mpH/min)
20 10 0 -10 OKT3 –
ns
100 50 NS PTENi
25 SA/CD3 20
G
DCA 2DG
15 10 5 0
0
50 100 150 Time (minutes)
50
SA/CD3
40
DCA
20 10 0
0
5
10
DCA 2DG
100 0
50 100 150 Time (minutes)
PMA
αCD3 1
SA/CD3
200
0
50 100 150 Time (minutes)
NS
WT NS WT CD3 Hif1af/fCd4Cre NS Hif1af/fCd4Cre CD3
300
2DG
30
J P
H
WT NS WT CD3 Hif1af/fCd4Cre NS Hif1af/fCd4Cre CD3
ECAR (%)
0
ECAR (mpH/min)
ECAR (mpH/min) ECAR (mpH/min) [ECARmax- ECARbasal]
E
WT NS WT CD3 Hif1af/fCd4Cre NS Hif1af/fCd4Cre CD3
F
I
150
P116
ECAR (mpH/min)
ECAR (mpH/min)
D
+
+
C39
1
5
10
PDHK1 (Y243) Total PDHK1
Figure S3. ZAP70 is required, but Akt, HIF1α, and calcium flux are dispensable for rapid activation-induced glycolysis (Supports Figure 4) A, ECAR trace of ZAP70-deficient (P116) or ZAP70-reconstituted (P116.CL39) Jurkat T cells stimulated with OKT3 crosslinked with αIgG. B, Tabulated results from multiple experiments as in A. C, Tabulated ECAR from multiple experiments of PA-R CD8+ T cells stimulated with streptavidin-crosslinked αCD3 at 3 μg/ml in the presence or absence of 0.5 mM EGTA and 10 nM thapsigargan. D, Tabulated ECAR from multiple experiments of PA-R CD8+ T cells in the presence or absence of 5 μM PTENi. E, Trace (left) and tabulated ECAR (right) of PA-R CD8+ T cells from Cd4Cre or Rictorf/f Cd4Cre mice stimulated with streptavidin cross-linked αCD3 at 3 μg/ml. F, Trace ECAR from naïve CD8+ T cells from Hif1af/f Cd4Cre or Hif1af/f stimulated with streptavidin-crosslinked αCD3 at 3 μg/ml. G, Trace ECAR from PA-R CD8+ T cells from Hif1af/f Cd4Cre or Hif1af/f stimulated as in F. H, Normalized ECAR from cells in G. I, Tabulated ECAR from multiple experiments of PA-R CD8+ T cells stimulated with streptavidin cross-linked αCD3 at 3 μg/ml or 33.3 ng/ml PMA. J, (Left) Immunoblot of indicated proteins in lysates of PA-R CD8+ T cells stimulated as in I for the indicated periods. (Right) tabulated densitometry results from multiple experiments. Results represent the mean of four (D, C, I) two (A, B, F, G, H) or three (E, J) experiments. * p < 0.05, ** p < 0.01, ***, p
