Immunity, Volume 51

Supplemental Information

Metabolic Profiling Using Stable Isotope Tracing

Reveals Distinct Patterns of Glucose Utilization

by Physiologically Activated CD8+ T Cells

Eric H.Ma,Mark J. Verway, RadiaM. Johnson, Dominic G. Roy, Mya Steadman, SebastianHayes, Kelsey S. Williams, Ryan D. Sheldon, Bozena Samborska, Penelope A.Kosinski, Hyeryun Kim, Takla Griss, Brandon Faubert, Stephanie A. Condotta, ConnieM. Krawczyk, Ralph J. DeBerardinis, Kelly M. Stewart, Martin J. Richer, VictorChubukov, Thomas P. Roddy, and Russell G. Jones

Supplementary Information for: Metabolic profiling using stable isotope tracing reveals distinct patterns of glucose

utilization by physiologically activated CD8+ T cells

Eric H. Ma, Mark J. Verway, Radia M. Johnson, Dominic G. Roy, Mya Steadman, Sebastian

Hayes, Kelsey S. Williams, Ryan D. Sheldon, Bozena Samborska, Penelope A. Kosinski, Hyeryun

Kim, Takla Griss, Brandon Faubert, Stephanie A. Condotta, Connie M. Krawczyk, Ralph J.

DeBerardinis, Kelly Marsh, Martin J. Richer, Victor Chubukov, Thomas Roddy, and Russell G.

Jones

Inventory of Supplementary Information:

Figure S1, Related to Figure 1. Physiologically activated CD8+ T cells display distinct

bioenergetic profiles.

Figure S2, Related to Figure 2. Proteomic and metabolic profiling reveals distinct metabolic

features of physiologically activated CD8+ T cells.

Figure S3, Related to Figure 3. Stable isotope infusion reveals glucose utilization patterns in

T cells in vivo.

Figure S4, Related to Figure 4. Pyruvate utilization by Teff cells differs in vivo.

Figure S5, Related to Figure 5. Glucose contributes to nucleotide and nucleotide sugar

biosynthesis in Teff cells in vivo.

Figure S6, Related to Figure 6. CD8+ Teff cell metabolism changes over the course of

infection.

Figure S7, Related to Figure 7. Glucose-dependent serine biosynthesis is essential for Teff

cell proliferation.

Figure S1, Related to Figure 1. Physiologically activated CD8+ T cells display distinct

bioenergetic profiles. (A) Left, representative flow cytometry plots for adoptively transferred

(CD45.2+) OT-I T cells (5 x 104; 2 x 106) into CD45.1 hosts and endogenous (Endo) Kb/OVA+

CD8+ T cells responding to LmOVA 3 dpi in the spleen. Right, percentage of OVA-specific CD8+

T cells in spleens of LmOVA-infected CD45.1 mice 3 dpi with 5 x 104 or 2 x 106 adoptively

transferred OT-I T cells. (B) Left, representative flow cytometry plots of IL-7R versus KLRG1

staining for Kb/OVA+ CD8+ T cells as in (A) from LmOVA-infected mice at 3 dpi. Right,

percentage of SLECs, MPECs, EECs, and DPECs of OVA-specific CD8+ T cells in spleens of

LmOVA infected CD45.1 mice 3 dpi. (C) Surface expression of CD69, CD25, CD44, and CD62L

for OT-I CD8+ Tn or Teff cells in vitro or in vivo 3 dpi. (D) Representative flow cytometry plots

of CD44 versus intracellular IFN-g for OT-I CD8+ Teff cells cultured in vitro or ex vivo 3 dpi

following re-stimulation with PMA and ionomycin. (E) Percentage of ATP generated from

glycolysis versus OXPHOS for CD8+ T cells cultured in vitro or ex vivo 3 dpi with LmOVA.

Seahorse analysis was conducted under normal cell culture media conditions (Glc, 25mM; Gln, 4

mM). The glycolytic index (GI) of T cells in each condition is shown. Data represents mean ± SD

for biological replicates (Tn in vitro, n = 20; Teff in vitro, n = 15; Tn ex vivo, n = 17; Teff ex vivo,

n = 14) (F) Bioenergetic capacity plot for CD8+ Tn cells cultured in vitro or ex vivo 3 dpi. The

rectangles define the maximum bioenergetic space of Tn cells as determined by the maximum

JATPox and JATPgly after treatment with FCCP and monensin, respectively. Points in the

bioenergetic space define the baseline JATPox and JATPgly for in vitro and ex vivo CD8+ Tn cells

(n = 17).

Figure S2, Related to Figure 2. Proteomic and metabolic profiling reveals distinct metabolic

features of physiologically activated CD8+ T cells. (A) Schematic of naïve and Thy1.1+ magnetic

bead isolation. Naïve Thy1.1+CD8+ OT-I T cells were adoptively transferred into C57BL/6 mice,

followed by LmOVA infection one day later. At 3 dpi, T cells were isolated from the spleen (see

STAR Methods). (B) Representative flow cytometry plots assessing the purity of T cells following

magnetic bead isolation. Thy1.1+ OT-1 CD8+ T cells were adoptively transferred into Thy1.2+

C57BL/6 mice by IV injection (2 x 106 cells/mouse), then Thy1.1+ (top) or naïve CD8+ (bottom)

T cells were isolated from LmOVA-infected mice 3 dpi. Numbers indicate cell purity at each step.

(C) Ratio of relative protein abundance (Teff/Tn) for glycolytic and TCA cycle enzymes for OT-

I CD8+ T cells cultured in vitro or ex vivo 3 dpi. Data represent mean ± SEM for biological

replicates (in vivo, n = 6; in vitro, n = 3). (D) Comparison of metabolite abundance in standard

harvesting (PBS wash) versus bead isolation (Bead-Isolation) conditions from in vitro cultured

Teff cells. (E) Relative abundance of metabolites (glycolytic, TCA cycle, and amino acids) in Teff

cells cultured in vitro following standard harvesting (PBS wash) or magnetic bead isolation

procedure. Relative metabolite abundance was determined by GCMS (n = 3). (F) Relative

abundance of specific metabolites in the isolation buffer prior to and after magnetic bead isolation

(Figure S2A).

LiverBlood

Spleen0

20

40

60

80

100

% o

f poo

l

m+1m+2m+3m+4m+5m+6

D-GlucoseA

Liver Spleen0

20

40

60

80

100

% o

f poo

l

Pyruvate

Liver Spleen05

101560708090

100

% o

f poo

l

CitrateB C

m+0 m+3 m+0

LiverBlood

Spleen0

5

1060708090

100

% o

f poo

l

Aspartate

m+0 m+2Liver

BloodSpleen

05

101560708090

100

% o

f poo

l

Glutamate

m+0 m+2

D

m+2

T cell 13C labeling (fraction of pool)

Sple

en 13

C la

belin

g (fr

actio

n of

poo

l)

UDP-Glc

UDP-Glc

GlyGMP

Ala

Citrate

FumarateMalate

Ser

AMP

TnTeff

UMP

SAMSAH

Figure S3 Ma et al.

E

Figure S3, Related to Figure 3. Stable isotope infusion reveals glucose utilization patterns

in T cells in vivo. (A) Mass isotopomer distribution (MID) of D-glucose for liver, blood, and

spleen of C57BL/6 mice following IV U-[13C]-glucose infusion. Data represent mean ± SD for

biological replicates (n = 7). (B–C) Labeling patterns of pyruvate (B) and citrate (C) in the liver

and spleen of mice following 120 minutes of U-[13C]-glucose IV infusion. Unlabeled (m+0) and

fully labeled isotopomers for pyruvate (m+3) are shown. For citrate, unlabeled (m+0) and

prominent label (m+2, pyruvate entry by PDC) isotopomers are shown. (D) Labelling patterns of

U-[13C]-glucose-derived aspartate and glutamate in liver, blood, and spleen following 120

minutes of U-[13C]-glucose IV infusion. Shown are the unlabeled (m+0) and prominent label

(m+2) isotopomers (n = 6). (E) Comparison of 13C-glucose-derived metabolites in the spleen

versus Tn or Teff cells following 120 min U-[13C]-glucose IV infusion in LmOVA-infected mice

3 dpi. Select metabolites and amino acids are shown.

Figure S4 Ma et al.

0

2

4

6

8

Rel

. Abu

ndan

ce

U-[13C]-Glc → Pyr (m+3)

- +IL-2

0.0

0.5

1.0

1.5

2.0

Rel

. Abu

ndan

ce

U-[13C]-Glc → Ala (m+3)

- +IL-2

0

5

10

15

Rel

. Abu

ndan

ce

U-[13C]-Glc → Lac (m+3)

- +IL-2

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of L

ac p

ool

U-[13C]-Glc → Lac

m+0

m+1

m+2

m+3

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of A

la p

ool

U-[13C]-Glc → Ala

m+0

m+1

m+2

m+3

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of P

yr p

ool

U-[13C]-Glc → Pyr

m+0

m+1

m+2

m+3

0

1

2

3

Rel

. Abu

ndan

ce

U-[13C]-Glc → Cit

- +IL-2

0.0

0.5

1.0

1.5

2.0

2.5

Rel

. Abu

ndan

ce

U-[13C]-Glc → Fum

- +IL-2

0

2

4

6

Rel

. Abu

ndan

ce

U-[13C]-Glc → Mal

m+2

m+3

- +IL-2

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of C

it po

ol

U-[13C]-Glc → Cit

m+0

m+1

m+2

m+3

m+4

m+5

m+6

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of F

um p

ool

U-[13C]-Glc → Fum

m+0

m+1

m+2

m+3

m+4

PBS was

h

Bead I

solat

ion0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of M

al p

ool

U-[13C]-Glc → Mal

m+0

m+1

m+2

m+3

m+4

A

B C

1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of p

ool

m+0m+1m+2m+3

U-[13C]-Glc → Lac

ex vi

voin vitro(days

post-activation)

Figure S4, Related to Figure 4. Pyruvate utilization by Teff cells differs in vivo. (A) MID of

U-[13C]-glucose-derived glycolytic (lactate, pyruvate, alanine) and TCA cycle (citrate, fumarate,

malate) metabolites in Teff cells 4 days post OVA-peptide activation in vitro. Metabolite extracts

were isolated from OT-I CD8+ Teff cells cultured with U-[13C]-glucose for 6 hours, followed by

standard extraction (PBS wash) or magnetic bead isolation (Bead Isolation). Relative metabolite

abundance was determined by GCMS. Data represent mean ± SEM for biological replicates (n =

3). (B) Fractional enrichment of U-[13C]-glucose-derived lactate from Figure 4E. (C) Relative

abundance of fully labeled (m+3) isotopomers of U-[13C]-glucose-derived lactate, pyruvate,

alanine, and major isotopomers (m+2,3) for U-[13C]-glucose-derived citrate, fumarate, and malate.

Metabolite extracts were isolated from OT-I CD8+ Teff cells activated in vitro and cultured with

U-[13C]-glucose for 2 hours in the presence or absence of 50U/mL IL-2. Relative metabolite

abundance was determined by GCMS (n = 3).

Figure S5, Related to Figure 5. Glucose contributes to nucleotide and nucleotide sugar

biosynthesis in Teff cells in vivo. (A) Relative abundance of UDP-glucose biosynthesis enzymes

in CD8+ OT-I Teff cells in vitro or in vivo 3 dpi. Protein abundance in Teff cells was normalized

to expression levels in Tn cells. Data represent mean ± SEM for biological replicates (in vitro, n =

3; in vivo, n = 6). (B) Left, relative abundance of unlabeled (12C, white) and 13C-glucose-labelled

(black) UDP-glucose and, right, MID of major 13C-glucose-derived isotopologues of UDP-glucose

in Tn and Teff cells cultured in vitro. (C) Relative abundance of UDP-GlcNAc biosynthesis

enzymes in CD8+ OT-I Teff cells in vitro or in vivo 3 dpi. Protein abundance in Teff cells was

normalized to expression levels in Tn cells (in vitro, n = 3; in vivo, n = 6). (D) Left, relative

abundance of unlabeled (12C) and 13C-glucose-labelled UDP-GlcNAc and, right, MID of major

13C-glucose-derived isotopologues of UDP-GlcNAc in Tn and Teff cells cultured in vitro. (E–F)

MID of U-[13C]-glucose-derived UMP (E) and ADP (F) for CD8+ T cells cultured in vitro or

analyzed by in vivo infusion 3 dpi.

62%

29% 6%

T cell 13C labeling (% of pool)

Sple

en 13

C la

belin

g (%

of p

ool) Tn 6dpi

Teff 6dpi

UDP-Glc (m+1,2,3)

UDP-Glc (m+6)

CD44 CD62L

A

F

B E

H

I

Lac (m+3) Ala (m+3)0.0

0.1

0.2

0.3

0.4

Rel

. Fra

ctio

nal E

nric

hmen

t(M

etab

olite

/Glc

m+6

) TnTeff

n.d.n.d.

3dpi

G

Glu (m+2) Gln (m+2)0.0

0.1

0.2

0.3

Rel

. Fra

ctio

nal E

nric

hmen

t(M

etab

olite

/Glc

m+6

)

TnTeff

3dpi

3dpi

TnTe

ff Tn

Teff 3d

pi

Teff 6d

pi0

500

1000

1500

2000

J ATP

tota

l (pm

ol A

TP/m

in)

in vitro ex vivo

JATP gly JATP ox

Teff6dpi

Teff3dpi

Teff6dpiTeff3dpi

CD44

IFNγ

C

D

Figure S6 Ma et al.

TnTe

ff Tn

Teff 3d

pi

Teff 6d

pi0

20

40

60

80

100

Supp

ly F

lexi

bilty

Inde

x (%

)

in vitro ex vivo

IL-7R

KLR

G1

Teff3dpi Teff6dpi

0

10

20

30

40

50

60

%G

ranz

yme

B+ (o

f CD

45.2

+ )

Teff3dpi Teff6dpi

0

10

20

30

40

50

60

70

%IF

N-γ

+ (o

f CD

45.2

+ )

45%

26% 26%

CD44

Gra

nzym

e B

Endo

OT-I: 5

x104

0

50

100

% o

f OVA

TET

+

SLECs

EECs

MPECs

DPECs

Teff6dpiTeff3dpi

59% 52%

45% 47%

OT-I: 5x104Endo6 dpi LmOVA

(gated on CD45.2+)

(gated on OVA TET+)

(gated on CD45.2+)

Figure S6, Related to Figure 6. CD8+ Teff cell metabolism changes over the course of

infection. (A) Representative flow cytometry plots of CD44 and CD62L expression for CD8+ OT-

I T cells isolated from LmOVA-infected mice at 3 or 6 dpi. (B) Left, representative flow cytometry

plots of CD44 versus intracellular IFN-g staining for adoptively transferred CD45.2+ OT-I CD8+

T cells isolated from spleens of CD45.1+ LmOVA-infected mice at 3 or 6 dpi. Splenocytes were

re-stimulated with OVA257 peptide. Right, percentage of IFN-g producing CD45.2+ T cells are

shown. Data represent mean ± SEM for biological replicates (n = 3). (C) Left, representative flow

cytometry plots of CD44 versus intracellular Granzyme B staining for adoptively transferred

CD45.2+ OT-I CD8+ T cells isolated from spleens of CD45.1+ LmOVA-infected mice at 3 or 6

dpi. Splenocytes were re-stimulated with OVA257 peptide. Right, percentage of Granzyme B

producing CD45.2+ T cells are shown (n = 3). (D) Left, representative flow cytometry plots of IL-

7R versus KLRG1 staining for endogenous Kb/OVA+ CD8+ T cells response and CD45.2+ OT-I

CD8+ T cells adoptively transferred into CD45.1 mice at 6 dpi with LmOVA. Right, percentage of

short-lived effector cells (SLECs), memory-precursor effector cells (MPECs), early effector cells

(EECs), and double-positive effector cells (DPECs) of OVA-specific CD8+ T cells in spleens of

LmOVA infected CD45.1 mice 6 dpi (n = 3). (E) ATP production rates for Tn and Teff CD8+ OT-

I T cells cultured in vitro or ex vivo 3 or 6 dpi under normal cell culture media conditions. JATPtotal

is the sum of the glycolytic (JATPgly) and OXPHOS (JATPox) ATP production rates (Tn in vitro, n

= 4; Teff in vitro, n = 11; Tn ex vivo n = 5; Teff3dpi, n = 12; Teff6dpi, n = 10). (F) Supply flexibility

index of Tn and Teff cells as in (Figure 6E) (Tn in vitro, n = 4; Teff in vitro, n = 11; Tn ex vivo n

= 5; Teff3dpi, n = 12; Teff6dpi, n = 10). (G–H) Fractional enrichment of U-[13C]-glucose-derived

metabolites in T cells at 6 dpi. Fractional enrichment of (G) lactate (m+3) and alanine (m+3) and

(H) glutamate (m+2) and glutamine (m+2) in Tn and Teff cells relative to U-[13C]-glucose levels

in spleen at 6 dpi. Red dashed line denotes fractional enrichment observed in Teff cells at 3 dpi (n

= 6). (I) Comparison of 13C-glucose-derived metabolites in the spleen versus Tn and Teff cells

following 120 min U-[13C]-glucose IV infusion in LmOVA-infected mice at 6 dpi. UDP-Glc

isotopomers are shown.

LiverBlood

Spleen0

5

1060708090

100%

of p

ool

m+0m+1m+2m+3

0

20

40

60

80

%IF

N-γ

+ (o

f Thy

1.1+ )

shPhgdhSer/Gly

- + - ++ + - -

0

1

2

3

4

#Thy

1.1+ I

FN-γ

+ (x

106 )

shPhgdhSer/Gly

- + - ++ + - -

6000

8000

10000

12000

14000

IFN

-γ M

FI

shPhgdhSer/Gly

- + - ++ + - -

shPhgdh

CD44

IFN

-γ+

(gated on Thy1.1+)

shPhgdhshCtrl+Ser/Gly Feed -Ser/Gly Feed

shCtrl

48%58% 41% 55%

LmOVA

+Ser/Gly feed

-Ser/Gly feed

-14 0 7-1

-14 0 7-1

CD8+ OT-I

OT-I T cell response(Thy1.1)

-Ser/Gly feed

-14 0 7-1

+Ser/Gly feed

-14 0 7-1

shCtrl

OT-I

shCtrl

shPhgdh

shPhgdh

C57BL6

*

******

E

G

PBS was

hBea

d

Isolat

ion

0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of p

ool

U-[13C]-Glc → Glycine

m+0

m+1

m+2

PBS was

hBea

d

Isolat

ion

0.0

0.2

0.4

0.6

0.8

1.0Fr

actio

n of

poo

l

U-[13C]-Glc → Serine

m+0

m+1

m+2

m+3

shCtrl (+Ser/Gly)

shCtrl (-Ser/Gly)

shPhgdh (+Ser/Gly)

shPhgdh (-Ser/Gly)

CD44 CD25

Tn Teff0

10

20

30

40

50

% o

f poo

l

13C6-Glc→Ser (in vitro)

Tn Teff0

10

20

30

40

50

% o

f poo

l

13C6-Glc→Gly (in vitro)

m+1m+2m+3

A

D

B

F

C

Figure S7 Ma et al.

PBS was

hBea

d

Isolat

ion

0.0

0.5

1.0

1.5

Rel

. Abu

ndan

ce

U-[13C]-Glc → Serine

C12

C13

PBS was

hBea

d

Isolat

ion

0.0

0.5

1.0

1.5

Rel

. Abu

ndan

ce

U-[13C]-Glc → Glycine

C12

C13

in vit

ro

ex vi

vo0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of p

ool

U-[13C]-Glc → Serine

m+0

m+1

m+2

m+3

in vit

ro

ex vi

vo0.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of p

ool

U-[13C]-Glc → Glycine

m+0

m+1

m+2

in vit

ro

ex vi

vo0.00

0.05

0.10

0.15

0.20

Rel

. Abu

ndan

ce

U-[13C]-Glc → Glycine

13C

12C

in vit

ro

ex vi

vo0.00

0.02

0.04

0.06

0.08

0.10

U-[13C]-Glc → Serine

Rel

. Abu

ndan

ce

13C

12C

Figure S7, Related to Figure 7. Glucose-dependent serine biosynthesis is essential for Teff

cell proliferation. (A) MID of U-[13C]-glucose-derived serine in the liver, blood, and spleen of

LmOVA-infected mice (3 dpi) following 120 minutes of U-[13C]-glucose IV infusion. Data

represent mean ± SEM for biological replicates (n = 6). (B) MID of U-[13C]-glucose-derived serine

and glycine for Tn and Teff cells generated in vitro. T cells were cultured for 6 hours in medium

containing U-[13C]-glucose (n = 3). (C) Top, relative abundance of unlabeled (12C) and 13C-

glucose-derived serine and glycine and, bottom, MID of U-[13C]-glucose-derived serine and

glycine in Teff cells cultured with U-[13C]-glucose for 2 hours in vitro or ex vivo 3 dpi. Relative

metabolite abundance and MID was determined by GCMS (n = 3). (D) Top, relative abundance of

unlabeled (12C) and 13C-glucose-labelled serine and glycine and, bottom, MID of U-[13C]-glucose-

derived serine and glycine in Teff cells cultured with U-[13C]-glucose for 6 hours in vitro.

CD8+OT-I T cells were activated in vitro and extracted for metabolites following standard

harvesting (PBS wash) or magnetic bead isolation procedure (Bead Isolation). Relative metabolite

abundance and MID was determined by GCMS (n = 3). (E) Representative flow cytometry plots

for CD44 and CD25 surface expression on Teff cells expressing control (shCtrl) or Phgdh-

targeting (shPhgdh) shRNAs cultured in full media or serine/glycine free media. (F) Schematic of

dietary intervention and T cell adoptive transfer and LmOVA infection system. (G) Response of

control and Phgdh-knockdown OT-1+ CD8+ T cells to LmOVA infection. Left, representative flow

cytometry plots of CD44 versus intracellular IFN-g staining for OVA-specific Thy1.1+/Thy1.2+

OT-1+ CD8+ T cells in the spleen of LmOVA-infected mice 7 dpi. Mice were maintained on control

(+) or serine/glycine-free (-) chow two weeks prior to adoptive transfer of Thy1.1+/Thy1.2+ OT-

1+ CD8+ T cells expressing shCtrl or shPhgdh hairpins and then were maintained on respective

diets for the duration of the infection. Right, percentage, total numbers, and MFI of OVA-specific

(Thy1.1+/Thy1.2+) CD8+ IFN-g+ T cells in spleens of LmOVA-infected mice 7 dpi (n = 5).