THE IMMUNITY-METABOLISM LINK

Immunology

Cell Metabolism Assays

Research

for

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XF Fatty Acid Oxidation Profile Exogenous & Endogenous Fatty Acid Oxidation

XF Glycolysis Stress Test Profile Glycolytic Function

051015202530354045

Extra

cellu

lar A

cidi

ficat

ion

Rate

(EC

AR)

(mpH

/min

)

Glycolysis

Glucose Oligomycin 2-DG

Glycolytic Capacity

Glycolytic Reserve

0 10 20 30 40 50 60 70 80 90 100

Non-glycolytic Acidification

Time (minutes)

XF Cell Mito Stress Test Profile Mitochondrial Respiration

Basal Respiration

FCCPRotenone & antimycin A

Proton Leak

004080120160200240280320360

10 20 30 40 50 60 70 80 90 100 110

Non-mitochondrial Respiration

Time (minutes)

Oxy

gen

Con

sum

ptio

n Ra

te (O

CR)

(p

mol

/min

)

Spare Capacity

ATPProduction

Oligomycin

Maximal Respiration

XF Cell Energy Phenotype Test Metabolic Phenotype & Potential

Oxy

gen

Con

sum

ptio

n Ra

te (O

CR)

Extracellular Acidification Rate (ECAR)

GOLD STANDARD METABOLIC ASSAYSMEASURING THE KEY PARAMETERS OF CELL METABOLISM

FCCPRotenone & antimycin A

020406080100120140160180200

10 20 30 40 50 60Time (minutes)

Oxy

gen

Con

sum

ptio

n Ra

te (O

CR)

(p

mol

/min

)

Oligomycin

Basal

Maximal

Exogenous Palmitate Oxidation

Endogenous FAO

Exogenous Palmitate Oxidation

Endogenous FAO

CELL LINEAGE AND IMMUNOMETABOLISM

Naive T cell

ActivatedT cell

E�ectorT cell

MemoryT cell

Fatty acid oxidation

Mitochondrial respiration (OCR)

Glycolysis (ECAR)

FUNCTIONAL XF METABOLIC ASSAYS - THE INTERSECTION OF IMMUNOLOGY & METABOLISM XF TECHNOLOGY PROVIDES THE TOOLS TO MEASURE IMMUNOMETABOLISM

Immunologists study how the innate and adaptive immune system recognizes and responds to insults. Whether the research focus is on a specific immune cell type, signaling pathway, or disease area, immunologists are actively exploring the mechanisms that drive and perpetuate antigen recognition and response.

Immune system metabolism (referred to as ‘immunometabolism’) has emerged as a key mechanism in understanding the connection between metabolic pathways and immune responses. Each immune cell type has a specialized role in an immune response, as well as a preferred metabolic pathway to generate energy required to maintain homeostasis.

Research into metabolic changes provides insight into metabolic signature, signaling, and substrate preference. These metabolic choices are leading to new opportunities to modulate immunological response for therapeutic intervention.

XF Technology is at the forefront, providing powerful and effective tools to explore the burgeoning field of immunometabolism.

Metabolism

Cell Type

SignalingPathways

DiseaseArea

GLUCOSE

GLUTAMATE

FATTY ACID

FATTY ACIDLACTATE CITRATE

Acetyl-CoA

TCA

GLUTAMINEGLYCOLYSIS

(ECAR)

MITOCHONDRIAL RESPIRATION

(OCR)

GLUTAMATE

ETC

PYRUVATE

SUBSTRATES AND METABOLISM

Extracellular Acidification Rate (ECAR)

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

Glycolysis

XFp Cell Energy Phenotype Profile

Mito

chon

dria

l Res

pira

tion

Metabolic Potential

Aerobic

Quiescent

BaselinePhenotype

StressedPhenotype

Energetic

Glycolytic

Rev

2

ImmunologyBrochure.indd 1-3 7/6/15 3:25 PM

Control

ns100

80

60

40

20

0

****

Rapa

- glucose

+ glucose

MEASURING THE KEY PARAMETERS OF IMMUNOMETABOLISM

METABOLIC SIGNATURES Cell activation, amplification, and effector function are crucial aspects of the immune cell life cycle. Researchers are using XF Technology to examine the cross-talk between immune cells and their metabolic signatures to gain insight into disease pathology, etiology, and possible treatment options. The XF Cell Mito Stress Test measures the key parameters of mitochondrial function: basal respiration, ATP-linked respiration, proton leak, maximal respiration, and spare respiratory capacity. The XF Glycolysis Stress Test measures the three key parameters of glycolytic function: glycolysis, glycolytic capacity, and glycolytic reserve.

SUBSTRATE UTILIZATION, FLEXIBILITY & DEPENDENCY Each immune cell type has a specific function within the immune system. Cell fate decisions, activation, amplification, and effector function are driven by metabolism. To maintain the energy demands of the cell at each stage of its life cycle, the metabolic requirements for substrates or fuels that feed into the metabolic ‘engines’ are altered. XF Technology provides the capability to examine the effect of substrate utilization and dependency, yielding powerful metabolic data.

XF Cell Mito Stress Test profiles metabolic signatures of activated bone marrow-derived macrophage subtypes.

Mouse bone-derived macrophages

Huang SC et al., (2014) Nat Immunol.

Human T cells

Hegedus A et al., (2014) Retrovirology

XF Assays reveal the role of glycolysis in HIV infection.

0

2

4

6

8

10

12

100 200 3000 400 500

Inhibitor or DMSOα-CD3 and α-CD28

PI(3)K/Akt inhibitorDMSO

Even

ts (%

max

)

eFluor 670

0100 104103102101

20

60

40

80

100

ActivatedActivated + PI(3)K inhibitor

0

125100755025

150175200225250275

0 30 60 75 10515 45 90 120

Oligomycin FCCP

SRC

Rotenone/ antimycin A

M0 M1 M2

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in)

XF Metabolic Switch Assay reveals distinct metabolic signatures in platelets and leukocytes.

Human platelets and leukocytes

Kramer PA et al., (2014) Redox Biol.

456789

10

3210

0 2 31 4 5 6 7 8 9 10

BasalOligomycinMonocytes

Platelets

Lymphocytes

Neutrophils

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in/μ

g pr

otei

n)

Extra

cellu

lar A

cidi

ficat

ion

Rat

e (E

CAR

) (%

mpH

/min

)

(mpH/min/μg protein) Extracellular Acidification Rate (ECAR)

GOLD STANDARD ASSAYS FOR MEASURING METABOLIC REPROGRAMMING

Human T cells

Yin Y et al., (2015) Sci Transl Med.

Murine T helper cells

Chatterjee S et al., (2014) Cancer Res.

METABOLIC EFFECT OF THERAPEUTICS The primary role of the immune system is to protect the host by targeting foreign antigens, controlling or clearing infections, and attacking cancerous cells. However, metabolic interventions present a largely untapped area of disease research. XF Technology provides the necessary tools to understand the effects of therapeutic candidates on the antigen of interest, and on the immune system.

Real-time activation reveals metabolic signatures of lupus patient-derived CD4+ T cells.

XF Assays reveal a metabolic switch which correlates to the antitumor ability of Th17IL1β cells.

Tum

or s

ize (m

m2 )

Days

400

500 Control

Th17TGFβ1

Th17IL1β

300

200

100

00 10 155 20 25

Don

or c

ells

(per

mL

of b

lood

)

3º Day 7Recall

1º Day 7Recall

0

1x105

2x105

3x105

4x105 *

Murine T cells

Fraser KA et al., (2013) Immunity

XF Assays reveal that tertiary immunization increases memory CD8+ T cell recall.

1000

750

500

250

00 20 40 60 80 100 120

Oligomycin FCCP Rotenone

3ºSRC

1ºSRC

Naive 1º Day 7 3º Day 7

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in)

Time (min)

80

60

40

Th17TGFβ1 Th17IL1β

20

0

2,000

1,500

1,000

500

0

**

***

Exrta

cellu

lar A

cici

ficat

ion

Rat

e (E

CAR

) (m

pH/m

in)

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

pmol

O2/m

in

THE WORLD’S MOST ADVANCED METABOLIC ANALYZERS

PROVEN TECHNOLOGY FOR CUTTING EDGE RESEARCH There are nearly 2,000 references utilizing XF Technology published in leading journals such as Nature and Cell. Scientists are embracing XF Technology to identify metabolic phenotypes and reprogramming to target metabolic pathways for therapeutic purposes.

Murine T cells

van der Windt GJ et al., (2013) PNAS

Murine T cells

Fletcher M et al., (2015) Cancer Res.

Human B cells

Liu T et al., (2014) Cell Death Dis.

I

II

III

IV

Lactate

Pyruvate

Glucose

Glycolysis ECAR (Extracellular Acidification Rate)

Mitochondrial Respiration OCR (Oxygen

Consumption Rate)

XF Assays reveal a role of fatty acid oxidation in memory T cell response.

XF Glycolysis Stress Test identifies L-Arginine dependence for proliferation in T cells.

XF Assays reveal a glucose dependence in B-cell acute lymphoblastic leukemia (B-ALL) cells.

XF Assays reveal glucose-insensitivity of naïve T cells in the presence of rapamycin.

400

300

200

100

00 10050 150

controlor

etomoxir

Control Etomoxir

PMA/ionomycin

Time (min)

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in)

No Fuel Glutamine Glutamine + Glucose

B-ALL

cell lin

e #1

B-ALL

cell lin

e #2

B-ALL

cell lin

e #3B ce

ll

line #1 B ce

ll

line #2 B ce

ll

line #3Ex

trace

llula

r Aci

dific

atio

n R

ate

(EC

AR)

(mpH

/min

)

METABOLIC REPROGRAMMING Signaling plays an important role in eliciting immunological response by coordinating immune cell communication and actions. Researchers are using XF Technology to probe the signaling and metabolic programming of immune cells.

XF Cell Mito Stress Test identifies IFNAR requirement for metabolic reprogramming.

Murine dendritic cells

Pantel A et al., (2014) PLoS Biol.

Human T cells

Gubser PM et al., (2013) Nat Immunol.

Real-time activation of memory T cells demonstrates a connection amongst signaling pathways, glycolysis, and proliferation.

XF Assays reveal Natural Killer (NK) cell dependence on mTORC1 following stimulation of IL-2 and IL-12.

Murine Natural Killer cells

Donnelly RP et al., (2014) J Immunol.

Uninfected HIV-infected

1000

800

400

600

200

030 5040 7060 80

Glucose Oligomycin

** **

300

200

17 85 9425 34 42 51 60 68 77

WT Poly ICIFNAR-/- Poly ICIFNAR-/- Poly PBS

WT PBS

100

00 8

FCCPOligomycin Rotenone/antimycin A

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in)

ECAR

(mpH

/min)

Rapa

Unstim

- -

IL2/12

40

20

*

+Extra

cellu

lar A

cidi

ficat

ion

Rat

e (E

CAR

) (m

pH/m

in)

Extra

cellu

lar A

cidi

ficat

ion

Rat

e (E

CAR

) (m

pH/m

in)

Time (min)Time (min)

5

10

15

20

0WT IFNAR -/-

ECAR

(mpH

/min)

60

αCD3/CD28

BaselineSRC

InducedSRC

Oligomycin

αCD3/CD28 Control

FCCP Rotenone/antimycin A

20

40

020 60 100

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

(pm

ol/m

in)

Glucose Oligomycin 2-DG

Peg-BSA Peg-Arg I

250

200

100

150

50

-50

02010 30 5040 7060 80 10090 110

% T

cell

s pr

olife

ratin

g(C

FSE)

(μg/ml pegylated protein)

100

75

50

25

00

0.50.25

0.125 1 2

***

Time (min) Time (min)Time (min)

Extra

cellu

lar A

cidi

ficat

ion

Rat

e (E

CAR

) (m

pH/m

in)

Time (min)

Time (min)

Murine T cells

Goldberg EL, et al., (2014) J Immol.

Extra

cellu

lar A

cidi

ficat

ion

Rat

e (E

CAR

) (m

pH/m

in)

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