part 1 of 3

“Measuring Antigen Specific T-cells using Surface and

Intracellular Staining Polychromatic Flow Cytometry”

3rd Annual CFAR Flow Cytometry Workshop6-10 May, 2013

Janet StaatsFlow Cytometry Core Facility

Center for AIDS ResearchDuke University Medical Center

E-mail: [email protected]

Part 1 of 3

Overview of PFC Assay

Duke University Medical Center

IL-4IL-2

TNFaIFNg

APC-T cellinteractions

Cytokine/Chemokineexpression

Rantes

Apoptosis

Proliferation/Death

Memory CD4 T Cell Response to Ag

From H. MaeckerDuke University Medical Center

CD4+

T cell cytokines

CD8+

CTL

APC MHCII CD4

CD8 cytokines

Ag

peptide

MHC I

T, B,or APC

MHC I

Wholeprotein

Optimalpeptide

Duke University Medical CenterFrom H. Maecker

Response to CMV pp65 Peptide Mix

0.19% 2.03%

pp65 protein peptide mix A2 peptide

1.14%

CMV lysate

0.87%

CD8

7.41%0.27% 0.04%0.27%

CD4

Duke University Medical CenterFrom H. Maecker

Peptide Mixes15 a.a.

11 a.a.

CMV pp65: pool of 138 peptidesHIV p55: pool of 120 peptides


Sampson Clinical Trial:11-Color Maturation/Function Panel

Basic Subset Markers:• CD3 (T-cells)• CD4 (T-Helper Subset)• CD8 (T-Suppressor Subset)

Exclusion Markers:• CD14 (Monocytes)• CD19 (B-cells)• vAmine (Dead cell marker)

Maturational Markers:• CD45RO• CD27• CD57

Functional Markers:• CD107• IFN-g• TNFa• IL-2


Wash

5. Permeabilize

Wash

6. IC Stain7. Acquisition

8. Analysis

Overview of 11-Color Assay

4. Lyse/Fix

BrefeldinMonensin

3. Surface Stain2. Stimulate

Wash

lymphocyteerythrocyte

cytokine

6 hrs

AmineCD14 CD3CD4CD8

CD45ROCD27CD57

IFNgIL2TNF

1. Thaw

Rest

CD1076 h

CostimSEB

CMVpp65

Wash

CD107 PE-Cy5

CD8+ CM Response

7+g+M+g+M+

M+

Monday Tuesday Wednesday Thursday - Friday


FSC-W

FSC

-H

88.3

<V705-A>: CD8 Q705

<G71

0-A

>: C

D4

CY

55P

E

57.8

36.3

0.79

FSC-A

SS

C-A

99.3

<Violet G-A>: CD3 Amcyan

<Vio

let H

-A>:

vA

min

e C

D14

PB

CD

19 P

B

41.4

Gating Strategy for 11-Color Maturation/Function Panel: 1 of 3

CD

4 Pe

rCP-

Cy5

.5

SSC

-A

Excl

usio

n (V

iole

t H)

FSC

-H

FSC-ACD3 AmCyanFSC-W

CD8 Alexa700

Ungated Singlets CD3+ Exclusion-

Scatter

Basic Gates:

CD4+CD8-

CD8+CD4-

CD4+CD8+

- 3 total


<G66

0-A

>: C

D27

CY

5PE

43 54.1

2.580.33

<G66

0-A

>: C

D27

CY

5PE

56.4 28.6

8.466.55

<V54

5-A

>: C

D57

Q54

5

0.12 1.07

55.942.9

<V54

5-A

>: C

D57

Q54

5

5.67 13.2

24.256.9

Gating Strategy for Sampson 11-Color Maturation/Function Panel: 2 of 3

<G66

0-A

>: C

D27

CY

5PE

22 62.5

11.73.98

<V54

5-A

>: C

D57

Q54

5

3.98 22.9

51.721.5

CD

57 F

ITC

CD

57 F

ITC

CD

57 F

ITCC

D27

APC

-Ale

xa75

0

CD

27 A

PC-A

lexa

750

CD

27 A

PC-A

lexa

750

CD45RO ECD

N

NN

CM

CMCMEM

EMEM

TE

TETE

E

EE

Maturational Gates:

CD4+CD8-

CD8+CD4-CD4+CD8+

CD45RO ECD

CD45RO ECD

Naive Central Memory

EffectorMemory

Terminal Effector Effector


EffectorMemory



EffectorMemory


- 5 per basic subset


<R710-A>: CD107a AX680

2.59CD107

Gating Strategy for Sampson 11-Color Maturation/Function Panel: 3 of 3

Functional & Boolean Gates: - 4 functional gates per maturational subset - 16 boolean gates per maturational subset

CM: CD8+CD4-

Boolean Gates

Polyfunctional (1: ++++)

Polyfunctional (4: +++)

Bifunctional (6: ++)

Monofunctional (4: +)

Nonfunctional (1: ----)

Key:7 = CD107g = IFN-g2 = IL-2T = TNF-a

1.14

IL-2

TNF-a

IFN-g

0.31

4.19


Visualizing PFC Data:CMVpp65-specific Polyfunctional Response in CD8+ Central Memory Subset Increases

Post-Vaccination

Betts, (2006) Blood 107, 4781-4789.Makedonas, (2006) Springer Semin. Immunopathol. 28, 209-219.

Simplified Presentation of Incredibly Complex Evaluations

Dr. Mario RoedererImmunotechnology SectionVRC / NIAID / NIH


Part 2 of 3

PFC Challenges


Challenges…

• Instrument - optical configuration, optimization, standardization, and calibration

• Reagent - optimization and standardization

• Sample processing• Staining protocols• Data Analysis - compensation &

gating• Operators• Volume of data (death-by-excel!)


Consistency across batchesCD38 vs HLA-DR Staining on Ctrl 5L

28Feb085L CD8+

04Marb085L CD8+

11Mar085L CD8+

06Mar085L CD8+


uncompensated

compensationFSC/SSC settings

PMT settings

highlow

Difficulties in doing Automated Analysis related to Instrument Settings

CD4

CD

3

IFNg

CD

69

CD4

SSC

FSC

SSC

optimal optimal


Challenges…




gating• Operators• Volume of data (death-by-excel!)


Optimization using Spillover Assessments: Using Titration Files to Assess Spreading Error

Violet G- CD3 AmCyan

<Blu

e B

-A>

<Vio

let H

-A>

<Red

C-A

>

<Red

B-A

>

Red

A-A

<Gre

en E

-A>

<Gre

en D

-A>

<Gre

en C

-A>

<Gre

en B

-A>

<Gre

en A

-A>

CD3AC (5ug/ml) Spillover assessment:

• After compensation CD3AC showed spilllover into Blue-B detector (FITC channel)

Blue Laser

Violet Laser

Red Laser

Green Laser

<Blu

e A

-A>

• Ottinger, et. al., Poster #28, 23rd Annual Clinical Cytometry Meeting (2008)• Mahnke, et. al. Clin Lab Med. 2007 September; 27(3): 469-v.• Lamoreaux, et. al., Nature Protocols 1, 1507-1516 (2006) on line 9 November 2006Duke University Medical Center

Spillover Assessments:CD3 AmCyan (5µg/mL) Spillover into CD27 (0.32µg/mL)

& CD57 FITC (1.8µg/mL)

• Spillover from CD3AC interferes with detection of dim CD27 pos cells

• Spillover from CD3AC does not

interfere with detection of CD57

• Spillover is acceptable if it does not interfere with proper classification of events

• mAb concentration may be varied to reduce spillover as long as frequency is unaffected

CD27 FITC

Blue B

SSC

CD3AmCyan

9.8e-4Unstained

SSC

0.047

Blue B

Unstained

66.3

4.58

0.13

CD57 FITC

CD3AmCyan

20.5


Is this positive???

CMV pp65 stimulated sample

Maecker, et. al.Duke University Medical Center

Tandems Degrade!

• Ice• Dark• Fix• Controls• 6 hours

Maecker, et. al.Duke University Medical Center

Challenges…




gating• Volume of data (death-by-excel!) Duke University Medical Center

9-Color Activation/Maturation Using Cryo-preserved PBMC


Batch Processing ErrorCD38 vs HLA-DR Staining on Ctrl 5L

28Feb085L CD8+Lot 05262

04Marb085L CD8+Lot 05262

11Mar085L CD8+Lot 05262

06Mar085L CD8+Lot 05262

26Feb085L CD8+Lot 05262


Challenges…




gating• Operator• Volume of data (death-by-excel!)


How would you gate?

Markers:CD3CD4CD8IL-2+IFNg(FSC)(SSC)


N CM EM TE E

Pre-Vaccination

33%

21%

27%

2%

17%

Post-Vaccination

8%

48%25%

2%

17%


Reproducible analysis allows us to measure an expansion of CD4+ CM cells post vaccination with

some degree of confidence

ICS Standardization Conclusions

• ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision (<20% C.V.), that improves as the frequency of responding cells increases.

• Gating is a significant source of variability, and can be reduced by centralized analysis and/or use of standardized gating.

• Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood.

• Use of pre-aliquoted lyophilized reagents for stimulation and staining can reduce variability.

BMC Immunology 2005, 6:13 http://www.biomedcentral.com/1471-2172/6/13 Duke University Medical Center

CIC ICS Gating Panel110 labs participated and there were 110 different approaches to gating

BeforeBackgate

AfterBackgate

IFNgBackgate

CD3 AmCyan

Excl

usio

n

0.38 5.74

CD4 Gated CD8 Gated

5.230.27

IFNg PE-Cy7

CD

4 Pe

rCP-

Cy5

.5

CD

8 A

PC-C

y7

BeforeBackgate

AfterBackgate

A

B

BACKGATING: purity & recovery


Gating bias in proficiency panel results

CD4 FITC

IL2+

IFNg

PE

Unstim CEF CMV pp65

0.02%

0.01%

0.16%

0.03%

0.02%

0.17%

0.02%

0.03%

0.21%


We NEED better analysis tools!!!Manual (Expert) vs. Automated Analysis of

4-Color ICS Data File (CMVpp65)

0.21%0.18%

CD4 FITC

1.9%1.65%

CD8 PerCP-Cy5.5

IFN

-g +

IL-2

PE

Expert GatingManual

Cluster GatingAutomated


Would you know a positive if you saw one?

Roederer. Cytometry Part A, 73A:384-385 (2008)Horton et. al. J Immuno Methods, 323:39-54 (2007)Maecker et. al. Cytometry Part A, 69A:1037-1042 (2006)Comin-Anduix et. al. Clin Cancer Res, 12(1):107-116 (2006)

2xSD?>0.05%? Outside

Normal RangeRCV?


Challenges…




gating• Operator• Volume of data (death-by-excel!)


Assay Complexity


Endpoints for 11-Color Maturation/Function Panel DEATH BY EXCEL ……..

Basic (3) Maturation (5) Function Boolean (16)CD4+ CD8-

CD4+ CD8+

CD4- CD8+

NaïveCentral MemoryEffector MemoryEffectorTerminal Effector

CD107IFN-gIL-2TNF-a

Basic (3) Maturation (5) Boolean (16)X X 240/stim=X 3 Stimulations/Sample (CoStim, SEB, CMVpp65) = 720 Endpoints/Sample

720 Endpoints/Sample x 200 Samples (192 Participants + 8 Controls) = 144,000 Endpoints/Trial

Note 1: Frequency of parent only, reporting units of #cells/µL doubles the total EP/trialDuke University Medical Center

Data Annotation - for all 143,280 data points!

Study IDMethodAssay NameBatch #OperatorSample IDVisit IDAccession #% Viable (Flow)% Viable (Guava)RecoveryCD4 countCD8 countGate Name (Parameter Names)Tube NameFile NameError Code (1-11)

Checking:X1 - for electronic dataX3 - for manual entry

Requires STRONG statistical support:• Quickly exceeds limits of excel• Format data for statistical analysis

• FJ: column (gates) vs row (file)• CSV: column (identifiers) vs row (single value)

• Check data• Manual check: 8sec/value x 143280 = 49 days!!!


Part 3 of 3Why does this matter??

Why are you here???


Why is Reproducibility Important?CFSE Standardization Results (13 EXPERT IM Labs):- Very high inter-laboratory variability.- High background in some laboratories.- Responses to Gag and Nef peptide pools were

detected in HIV negative (control) donors!

Example Gag stimulationHIV negative donor

Example CMVpp65 stimulationCMV positive donor

% C

D8+

CFS

E lo

w

LaboratoryDuke University Medical Center

History of Flow-based Proficiency/Standardization Efforts


The number of measurements outside the optimal range established by the GS was determined for each laboratory. Each laboratory performed a total of 54 measurements (27 for CD4+ cells and 27 for CD8+ cells). The red line represents 50% (=27) of the total measurements. Laboratories above this line had over 50% of their measurements outside the optimal range. The green line represents 20% of total measurements. The laboratories below this line had over 80% of their measurements within the optimal range.

ICS Proficiency Testing Results: March 2007


DAIDS ICS Proficiency:Round 6, 26Jun09 (CMVpp65)

CD4-CD8+ CD4+CD8-

IFNg

+ IL-

2 PE

CD3 APC-Cy7

Rep #1

Rep #2

Rep #3


Acknowledgements


Duke CFARKent WeinholdJennifer EnzorTwan WeaverJianling ShiCliburn Chan

Patricia D’Souza (DAIDS) CFSE Standardization:

Claire Laundry (NIML)

EQAPOL

Duke Tisch Brain Tumor CenterGary ArcherDuane MitchellJohn Sampson

CHAVI

VRCSteve PerfettoLaurie LamoureauxMario Roederer

CVCSylvia Janetski

Duke DTRIScottie Sparks (Roche)

part 1 of 3

Documents

cd4 cd8cd8 cd4cd4 cd8

cd8 responses

specific t cells

cytokine flow cytometry

cd8 cfc responses

hours of t cell stimulation

maecker4stimulating

cd4 cy55pe