The Use of Flow Cytometry in Clinical and Basic HIV-1...

Presented at the 4th INTEREST Workshop25-28 May 2010, Maputo Mozambique

The Use of Flow The Use of Flow CytometryCytometry in in Clinical and Basic HIVClinical and Basic HIV--1 Research1 Research

4th INTEREST Workshop

Maputo, Mozambique

25-28 June 2010

Guido Ferrari, MDe-mail: [email protected]

Duke University Medical CenterCenter for AIDS Research (CFAR)

International AIDS Training Research Program (IATRP)Duke Global Health Institute (DGHI)


Flow Flow CytometryCytometry is a Quantitative Method is a Quantitative Method That Measures Single Events (Cells)That Measures Single Events (Cells)

Light Amplification by Stimulated Emission of Radiation

Lasers generate intense beams of coherent light.

www.bdbiosciences.com/immunocytometry_systems/support/training/online


Basic Aspect of a Flow Basic Aspect of a Flow CytometerCytometer

Analog Digital


Flow Flow CytometryCytometry: A Quantitative Assay Platform: A Quantitative Assay Platform

Level of Expression

Freq

uenc

y of

the

Cel

lula

r Sub

set

0

100


The AchillesThe Achilles’’ Heel of Flow Heel of Flow CytometryCytometry: : Signal Spillover Signal Spillover

Non Compensated

Compensated

+ ++

-+--

Parameter 2

Para

met

er 1

+- ++

-+--

+

-

-

+-


What Can We Learn by Flow?What Can We Learn by Flow?• Frequency of Cell Populations and their

Functions1. At the subset level with fluorophores linked to Ab to perform surface

and intracellular staining.2. At the epitope specific cellular level: fluorophores linked to Tetramer

• Proliferative Capacity of the CellsBy using fluorescent compounds CFSE for lymphoproliferation assay

(LPA)

• Intracellular Expression of Proteins or Infectious Agents

1. GFP expressed cloned in cell lines and infectious agents2. Activated peptides (apoptotic mechanisms and killing ability)


Flow Flow CytometryCytometry Applications Applications in HIVin HIV--1 Infection1 Infection

• Clinical Applications

• Research Applications


Clinical Application: Patient ManagementClinical Application: Patient Management1 Assay for CD4 Count and Rx Adherence1 Assay for CD4 Count and Rx Adherence

Glencross et al. “CD8/CD38 activation yields important clinical information of effective antiretroviral therapy”. Cytometry (2008) vol. 74B (S1) pp. S131-S140

CD4 Count:

Clinical Stage CD8 Activation Virus Load

Virus Load Rx Adherence

CD8 Activation Rx Adherence


Please see Posters

• P_26: Coetzee L. et al.: “Monitoring CD38 activation in HIV-1 infected pts on Rx is a potentially cost-effective alternative to repeated VL testing”

• P_27: Pillay K. et al.: “Extended window period for CD38 activation marker testing to 48 hours post venesection”


Flow Flow CytometryCytometry Applications in HIVApplications in HIV--1 Pathogenesis1 Pathogenesis

• Clinical Applications

• Research Applications:1. Frequency of Immune Cellular Subsets (T, B, NK, DC subsets);

2. Memory Phenotype and Maturation Stage of Cellular Susbsets;3. Cellular Functions: Cytokine production, Cytotoxic Activity, Ab

production;

4. Proliferative Capability of Ag-specific and non-Ag-specific Cells.


405nm Violet Laser

(50mW, Diode)

Duke LSR II

QDot

705

QDot

605

QDot

545

QDot

655

QDot

565705/7

0

585/42660/40

605/40

560/

40

670LP

595LP

570LP

630LP

557LP

560/40535

LP

Am

Cyan

515/20 505LP

450/

50 Cascade BluevAminePacBluePE

Cy7

PE

Cy5

780/4

0

610/20710/50

660/40

575/2

5

740LP

640LP

600LP

690LP

532nm Green Laser

(150mW, Solid State)Duke LSRII

635nm Red Laser

(25mW, Diode)Duke LSRII

710/50685LP

780/60

740LP660/20

Alexa 680Alexa 700

APC-Cy7 APC-HL750Alexa 750

APC Alexa 647

PerCP-Cy5.5PE Cy5.5

PE-TRAlexa 610-PE

PE

Green

Duke LSRII Rm 230488nm Blue Laser (20mW, Solid State)

515/20505L

P

710/40

685LP

488LP

FITCCFSE GFP Calcein

PE

Blue

SSC

Qdot 545PacOrange

QDot

585

PerCP-Cy5.5

Detection of 8 parameters to identify all possibility for 4 combDetection of 8 parameters to identify all possibility for 4 combined parameters:ined parameters:44--color color CytometerCytometer = 25 tubes = 25 million cells = 25 ml of blood= 25 tubes = 25 million cells = 25 ml of blood88--color color CytometerCytometer = 1 tube = 1 million cell = 1 ml of blood= 1 tube = 1 million cell = 1 ml of blood


1. Stimulate

+ Brefeldin A

Wash

3. Permeabilize

Wash

4. Stain

Wash

5. Acquisition6. Analysis

6 h

cytokine

lymphocyte

erythrocyte

ICS Methodology ICS Methodology

EDTA

......

IFN-

+Brefeldin A

+-IFN-FITC

+ -CD8 PE

Ag presenting cell

T cell

Ag

......

+ -CD3 APC

2. Viability&SurfaceStaining

Horton et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. Journal of Immunological Methods (2007) vol. 323 (1) pp. 39-54


Gating Strategy for the ICS Assay AnalysisGating Strategy for the ICS Assay Analysis

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

77.9

2.17

CD

107a

IFN

- IL-2

MIP

-1

CD

107a

IFN TNF

CD

27

CD

27

CD

57

CD45R0

CD8+ Memory

memory CD8 Functions

Live Lymphocytes Subsets

CD

4

SSC

-A

Viab

ility

CD8FSC-ACD3

CD8+

FSC

-H

FSC-A

Singlets

0

10 2

10 3

10 4

10 562.7

Naive

Memory37.3

0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

38.8

Live CD3+

0

10 2

10 3

10 4

10 5

51.7

CD27+

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

2.42

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 50 10 2 10 3 10 4 10 5

97.7

Lymphocytes

0 50K 100K 150K 200K 250K

0

50K

100K

150K

200K

250K

51

45.6

CD57+

CD45RO+0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

0.071

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

49.7

39.5

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

IL-2

5.23

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 5

0 10 2 10 3 10 4 10 5

0.71

I.

CD45R0 CD45R0

TNF TNF TNF


Boolean Logic in Flow Boolean Logic in Flow CytometryCytometryThe Boolean logic in Flow Cytometry allows to identify populations that fell in different gates according to the “And”, “Or”, or “Not” principles.

Sub

set 1

“Not

”2

Subset 1“And”2


MultiparameterMultiparameter ICS to Identify Functional CD8ICS to Identify Functional CD8++ Memory Memory SubsetsSubsets

CD107+ TNF-+CD107+ IFN-+ IL-2+ MIP-1+ TNF-+

Boolean AnalysisCD107a

IFNIL2

MIP1TNF

Pies

+++++

++++-

+++-+

++-++

+-+++

-++++

+++--

++-+-

++--+

+-++-

+-+-+

+--++

-+++-

-++-+

-+-++

--+++

++---

+-+--

+--+-

+---+

-++--

-+-+-

-+--+

--++-

--+-+

---++

+----

-+---

--+--

---+-

----+

Functional Family

5+ 4+ 3+ 2+

II.

IFN+ IL2+ MIP-1+

Boolean Analysisor or or or

Total Functional CD8 Response by Memory Phenotype

CD

27

CD

57

CD45RO

Combined Analysis =

III.

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

2.17

CD

107a

IFN

-

IL-2

MIP

-1

CD

107a

IFN TNF

memory CD8 Functions

0102

103

104

105 2.42

0102

103

104

105

0 102 103 104 1050 102 103 104 105

0.071

0102

103

104

105

0 102 103 104 105

IL-2

5.23

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0.71

TNF TNF TNF

CM

EM

TE1+


Uniform Expansion of Activation markers in ED, LD and Effector antigen-specific CD4+ T-cells

Maenetje P, G, Gray CM, et al. J Immunol. 2010;184(9):4926-35.


Flow Flow CytometersCytometers in Africa by just 1 in Africa by just 1 companycompany

252

10 4

10Flow Flow CytometryCytometry does not have to be highly complex and does not have to be highly complex and can be implemented in researchcan be implemented in research--limited setting if focused limited setting if focused and aimed to study any of the parameters thus far and aimed to study any of the parameters thus far presented.presented.

3


1

8 2

1 8

3

1

22

12

3

6

10

26

Participants

• We received 101 applications from the countries indicated on the map.

• Twenty-four (24) applicants were selected for the symposia and 12 more for the African Flow Cytometry Workshops that followed the Symposium.

1


Organizing Committee *Scientific Committee and Faculty *

**

*

**

**

****

**


2009 Infectious Diseases in Africa: Measurement of Immune Respon2009 Infectious Diseases in Africa: Measurement of Immune Responsesses&&

3rd African Flow 3rd African Flow CytometryCytometry WorkshopWorkshop

NICD, Johannesburg 11NICD, Johannesburg 11--17 November 200717 November 2007



2009 Infectious Diseases in Africa: 2009 Infectious Diseases in Africa: Measurement of Immune ResponsesMeasurement of Immune Responses

Visit: Visit: http://www.immunopaedia.org.za/

Topics of the Symposium:

1. Faculty Seminars on Adaptive and Innate Immune Responses to HIV,Malaria, and TB;

2. 12 Presentations by Young Investigators on HIV, Malaria, and TB;

3. Workshops on writing research proposal.


2009 Infectious Diseases in Africa: Measurement of Immune 2009 Infectious Diseases in Africa: Measurement of Immune ResponsesResponses

&&3rd African Flow 3rd African Flow CytometryCytometry WorkshopWorkshop


Topics of the Workshop:

1. Instrument optimization for BD Calibur and LSR II

2. Panel Optimization

3. Assay standardization

4. Software training for DIVA, FlowJo, PESTLE, and SPICE


Infectious Diseases in Africa: Measurement of Immune ResponsesInfectious Diseases in Africa: Measurement of Immune Responses&&

3rd African Flow 3rd African Flow CytometryCytometry WorkshopWorkshop


Evaluation of the event’s impact:

1. Daily questionnaire reviewing the topic of the day. Qs&As were discussed by different groups the following day (8 attendees with 2-3 faculty);

2. Final questionnaire to review all of the topics with scores;

3. The scores were used to identify the recipients of two fellowships to attend an International Meeting or Technical Workshop.


Co-organizer of IDA and Workshop

Clive Gray (NICD, South Africa)

The Faculty and the Participants.

ACKNOWLEDGMENTSACKNOWLEDGMENTS

Sponsored by: African AIDS Vaccine Initiative; National Institute of Health; World Health Organization.

Supported by: National Institute Communicable Disease (NICD); Duke University CFAR, IATRP, and GHI; BD Bioscience; TreeStar Inc.; InVitrogen.

The Members of the Organizing and Scientific Committees.

The Use of Flow Cytometry in Clinical and Basic HIV-1...

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