Amina Kariminia Ph.D. Oncology Lab, Child & Family Research Institute, Department of Pediatrics,...

Amina Kariminia Ph.D.

Oncology Lab, Child & Family Research Institute,Department of Pediatrics, Faculty of Medicine

University of British Columbia

ICIA 2014 Milad Tower, Tehran

Outlines• A glance to multi-color flow cytometry

• Tregs in whole blood sample

• Kinetic of NFkB phosphorylation in pre B-cell leukemia

• Monitoring immuno-suprressor drugs

• Multi-color flow cytometry for normal human B-cell sub-populations

• MIFlowCyt

• Introducing Cy-TOF: mass-cytometry

Types of controlsTypes of controls

• Instrument set up control– PMT voltage settings– Compensation

• Gating controls– Isotype controls– FMO controls

• Biological controls– Unstimulated samples– Healthy donors

Fluorescence Minus One is not always an ideal controlFluorescence Minus One is not always an ideal control

FMO FMO quadrant copy/pasted CD4- quadrant setting

CD3

Gal-Ser tetramer

No FRET with proper panel of conjugated Abs

No FRET with proper panel of conjugated Abs

Flow cytometry of rare populations Flow cytometry of rare populations

Minimal Residual Disease

GFP

PBL stem cells

BM stem cells

Tetramers: Ag specific T cells

Minimal Residual Disease

GFP

PBL stem cells

BM stem cells

Tetramers: Ag specific T cells

Rare population & Flow cytometryPractical Considerations

Rare population & Flow cytometryPractical Considerations

Number of events should be increased for statistical accuracy 200,000-1,000,000

Proper controls

Proper instrument setting

Number of events should be increased for statistical accuracy 200,000-1,000,000

Proper controls

Proper instrument setting

Intracellular stainingIntracellular staining

Cytokine

&

Phospho protein

Cytokine

&

Phospho protein

Intracellular stainingIntracellular staining• Cytokines

Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection

• Cytokines

Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection

Intracellular stainingIntracellular staining

• TH1/2/17

Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired

• TH1/2/17

Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired

CD3

CD4

IFN-g

IL-17

IFN-g

IL-4

Phospho-protein detectionPhospho-protein detection

• Phospho-protein

Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection

• Phospho-protein

Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection

B-cell Acute lymphoblastic leukemia cell linesB-cell Acute lymphoblastic leukemia cell lines

• Phospho-protein

TLR2 ligand Pam2CSK

Pre B-cell leukemia cell lines

Phosphorylation of NFkB & degradation of IkB

• Phospho-protein

TLR2 ligand Pam2CSK

Pre B-cell leukemia cell lines

Phosphorylation of NFkB & degradation of IkB

Time 0

Time 20Time 10

Time 5

380

0

5

10

15

20

25

30

0 0.01 0.1 1 10

TLR-2 agonist ug/ml

% of pNFkB+IKBa-

Pam2

Pam3

OP-1

0

5

10

15

20

25

30

35

40

45

0 0.01 0.1 1 10

TLR-2 agonist

% of pNFkB+IKBa-

Pam2

Pam3

TLR1

TLR-2

TLR-6

TLR1

TLR-2

TLR-6

TLR1

TLR-2

TLR-6

Dose response curve and phospho-protein

kinetic analysis

Dose response curve and phospho-protein

kinetic analysis

B-cell acute lymphoblastic leukemia: IL-7R expression

B-cell acute lymphoblastic leukemia: IL-7R expression

CD19+CD10+

0 102 103 104 105

<PE-A>

0

102

103

104

105

Sample %

Specimen_001_379-CD127.fcs 10.7

Specimen_001_1279-CD127.fcs 3.14

IL-7Ra

Ig Light chainHealthy control

B-ALL relapse

YB-1YB-1

Interruption of host immune system recognitionBy HLA-DO up-regulation/decreasing

Ag-presentation Prevention of apoptosis by Survivin

Blocking the effect of cytotoxic drugsThrough IL-7 signaling and multi-drug resistance -1 (MDR-1) up-regulation

YB-1 in acute lymphoblastic leukemia YB-1 in acute lymphoblastic leukemia

Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1

Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1

0 102 103 104 105

<PE-Cy7-A>

0

102

103

104

105 0.0141 0.243

94.65.09

0 102 103 104 105<PE-Cy7-A>

0

102

103

104

105 0 0.0237

94.85.17

CD19

pYB-1 No IL-7 IL-7 stimulated

Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647

Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647

Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube

Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube

0 102 103 104 105

<Qdot 800-A>: CD27

0

102

103

104

105

0 102 103 104 105

<APC-A>: CD10

0

102

103

104

105

51.5

11.8

6.98

0 102 103 104 105

<V500-A>: IgD

0

102

103

104

105

0 102 103 104 105

<Qdot 800-A>: CD27

0

102

103

104

105

B-cell ImmunophenotypingB-cell Immunophenotyping

0 102 103 104 105

PE-A

0

102

103

104

105

29.9

0 102 103 104 105

PE-A

0

102

103

104

105

0 102 103 104 105

APC-A

0

5

10

15

20

25

CD27-PE

IgD-FITC

CD19+ (Pacific Blue) gated cells

CD10-APC

IgD-FITC

CD27-PE

Bone marrow Peripheral Blood

Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450

Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450

0 102 103 104 105

<V450-A>: FOXP3

0

102

103

104

105

0 102 103 104 105

<V450-A>: FOXP3

0

102

103

104

105

Cyto-Chex tube EDTA tubeCD3+CD4+ gated T cells

0 102 103 104 105

<Alexa 647-A>

0

102

103

104

105

4.26

0 102 103 104 105

<Alexa 647-A>

0

102

103

104

105

1.82

FOXP3-AF647

CD25-PE

Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647

Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647

HD IPEX

0 102 103 104 105

<Pacific Blue-A>

0

102

103

104

105

0 102 103 104 105

<Pacific Blue-A>

0

102

103

104

105

CD25-PE

CD127-BV421

HD IPEX

Immuno-supressor Monitoringdetection of surface CD69 up-regulation

upon CD2 activation (IPEX patient)

Immuno-supressor Monitoringdetection of surface CD69 up-regulation

upon CD2 activation (IPEX patient)

CD3+CD4+ gated T cells

Un-stimulated

Corticosteroid Sirolimus

0 102 103 104 105

V450-A: FOXP3

0

102

103

104

105 7.04 1.07

17.374.5

0 102 103 104 105

V450-A: FOXP3

0

102

103

104

105 31.1 5.62

13.849.4

0 102 103 104 105

V450-A: FOXP3

0

102

103

104

105 11.1 2.31

10.576.1

Stimulated

The Minimum Information about Flow cytometry Experiment

(MIFlowCyt)

The Minimum Information about Flow cytometry Experiment

(MIFlowCyt)

Ref: Cytometry part A

73A:926-030

2008

Ref: Cytometry part A

73A:926-030

2008

MIFlowCytMIFlowCyt

Simplify cross-platform comparison and integration,

Simplify cross-platform comparison and integration,

MIFlowCytMIFlowCyt• Experimental overview:

– Purpose/goal hypothesis

– Experiment variables

– Conclusions

– Quality control

MIFlowCytMIFlowCyt

• Flow sample (specimen):

– Material

– Source/organism/location

– Treatment

– Reagent/analyte/detector/reporter

MIFlowCytData presentation

MIFlowCytData presentation

• Experimental and Sample Information:

– Experimental design

– Sample preparation reagents

– Fluorescent reagents• Vendor• Catalog number• Clone

MIFlowCytData presentation

MIFlowCytData presentation

• Data acquisition:

– Instrument configuration

– Instrument identification

– Fluidics configuration

– Optical configuration

– Electronic configuration

MIFlowCytData presentation

MIFlowCytData presentation

• Data presentation:

– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious

– Total events

MIFlowCytData presentation

MIFlowCytData presentation

• Data presentation:

– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious

– Total events

CyTOF: mass -cytometry

Bendall et al, Science, 2011

Child & Family Research InstituteChild & Family Research Institute