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Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
The Use of Flow Cytometry To Study Acute HIV-1 Infection
5th INTEREST Workshop
Dar es Salaam, Tanzania
10-13 May 2011
Guido Ferrari, MD
Duke University Medical CenterCenter for AIDS Research (CFAR)
Duke Human Vaccine Institute (DHVI)International AIDS Training Research Program (IATRP)
Duke Global Health Institute (DGHI)
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Flow Cytometry is a Quantitative Method 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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Basic Aspect of a Flow Cytometer
Analog Digital
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Flow Cytometry: A Quantitative Assay Platform
Level of Expression
Freq
uenc
y of
the
Cel
lula
r Sub
set
0
100
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
The Achilles’ Heel of Flow Cytometry: Signal Spillover
Non Compensated
Compensated
+ ++
-+--
Parameter 2Pa
ram
eter
1
+- ++
-+--
+
-
-
+-
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
The Achilles’ Heel of Flow Cytometry: Signal Spillover
Non Compensated
Compensated
+ ++
-+--
Parameter 2Pa
ram
eter
1
+- ++
-+--
+
-
-
+-
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
The Achilles’ Heel of Flow Cytometry: Signal Spillover
Non Compensated
Compensated
+ ++
-+--
Parameter 2Pa
ram
eter
1
+- ++
-+--
+
-
-
+-
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Flow Cytometry Applications in Acute HIV-1 Infection
• Clinical Applications
• Research Applications
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Clinical Application: Patient Management1 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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Please see Posters
• P_10: Moodley K. et al.: “CD38 Activation (MFI) Assessment by Flow Cytometry in HIV+ and HIV- Patients with or without TB Co-Infection”
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
What is the level of CD8 activation during acute HIV-1 infection?
How does early initiation of HAART impact the activation of CD8+ T cells?
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Gay C., Ferrari G., et al. AIDS (2011) vol. 25 (7) pp. 941-949
% o
f par
enta
l CD
3+
Time from infection
Levels of Total CD8 T cells Activation during Acute HIV-1 Infection (n=61)
Does not Reset Following Initiation of HAART
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Flow Cytometry Applications in Acute HIV-1 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.
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Basic Aspect of a Flow Cytometer
Analog Digital
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
405nm Violet Laser
(50mW, Diode)
Duke LSR II
QDot
705
QDot
605
QDot
545
QDot
655
QDot
565
660/40
535LP
Am
Cyan
Cascade BluevAminePacBlue
PE
Cy7
PE
Cy5
710/50
532nm Green Laser
(150mW, Solid State)
Duke LSRII
635nm Red Laser
(25mW, Diode)Duke LSRII
710/50
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)
515/20
FITCCFSE GFP Calcein
PE
Blue
SSC
Qdot 545PacOrange
QDot
585
PerCP-Cy5.5
Detection of 8 parameters to identify all possibility for 4 combined parameters:4-color Cytometer = 25 tubes = 25 million cells = 25 ml of blood8-color Cytometer = 1 tube = 1 million cell = 1 ml of blood
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
1. Stimulate
+ Brefeldin A
Wash
3. Permeabilize
Wash
4. Stain
Wash
5. Acquisition6. Analysis
6 h
cytokine
lymphocyte
erythrocyte
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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Gating 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
57CD45R0
CD8+ Memory
memory CD8 Functions
Live Lymphocytes Subsets
CD
4
SS
C-A
Via
bilit
yCD8FSC-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 250K0
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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
What is the level of Ag-specific CD4 and CD8 activation during acute HIV-
1 infection?
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Riou C., Gray C. et al. submitted
Antigen-specific CD4 cells were significantly more activated, as perCD38 expression, when compared to the total memory compartmentin both acute and chronic infection
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Multiparameter ICS to Identify Functional CD8+
Memory Subsets
0.071
0
10 2
10 3
10 4
10 5
0 10 2 10 3 10 4 10 5
2.17
CD
107a
IFN
- IL-2
MIP
-1
CD
107a
IFN TNF
memory CD8 Functions
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
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
TNF TNF TNF
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Boolean Logic in Flow CytometryThe 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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Multiparameter ICS to Identify Functional CD8+
Memory Subsets
0.071
0
10 2
10 3
10 4
10 5
0 10 2 10 3 10 4 10 5
2.17
CD
107a
IFN
- IL-2
MIP
-1
CD
107a
IFN TNF
memory CD8 Functions
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
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
TNF TNF TNF
CD107+ IFN-+ IL-2+ MIP-1+ TNF-+
Boolean Analysis
CD107a
IFN
IL2
MIP1
TNF
Pies
++
+
+
+
++
++
-
++
+-
+
++
-+
+
+-
++
+
-+
+
+
+
++
+-
-
++
-+
-
++
--
+
+-
++
-
+-
+-
+
+-
-+
+
-+
++
-
-+
+-
+
-+
-+
+
--
+
+
+
++
--
-
+-
+-
-
+-
-+
-
+-
--
+
-+
+-
-
-+
-+
-
-+
--
+
--
++
-
--
+-
+
--
-
+
+
+-
--
-
-+
--
-
--
+-
-
--
-+
-
--
-
-
+
Functional Family
5+ 4+ 3+ 2+ 1+
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
CD4+ Responses
CD8+ Responses
Week 54-78Week 28-34Week 12-16Week 4-8Week 1-3
0.99 1.24 0.54 0.36 0.79
Freq.Funct.
Freq.Funct.
0.42 1.60 1.87 2.23 1.52
Comparison of Ontogeny of Functional CD4+ and CD8+ Memory T Cell Responses following Acute HIV Infection
G. Ferrari, Gray CM, et al. Unpublished Data
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
What functionality is displayed by the Ag-specific CD8 driving escape during acute HIV-1 infection?
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
We ranked the mapped epitopes within each patients according to the order of appearance of escape mutants
Ferrari G, Korber B. et al. PLoS Pathog (2011) vol. 7 (2) pp. e1001273
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
MIP-1production dominates early responses…
Early-M Late-M Non-M Early-M Late-M Non-M
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Frequency of Epitope-specific MIP-1Functional CD8+ T cells Correlates with order of Appearance of
Escape Mutants
Earliest Positive ICSTotal Freq Fxn 1 Fxn 2-5 MIP-1
p = 0.03 0.11 0.07 0.006
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Flow Cytometers in Africa by just 1 company
252
10 4
10Flow Cytometry does not have to be highly complex andcan be implemented in research-limited setting if focusedand aimed to study any of the parameters thus farpresented.
3
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
What is needed to maximize the utilization of Flow Cytometry in
limited resource setting?
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Way forward:1. To mentor young scientists
2. Create a network of young scientists3. To implement education of clinical staff
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
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
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Organizing Committee *Scientific Committee and Faculty *
**
*
**
**
****
**
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
2009 Infectious Diseases in Africa: Measurement of Immune Responses&
3rd African Flow Cytometry Workshop
NICD, Johannesburg 11-17 November 2007
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
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; 2 will be re-invited from the previous event because were the top performers
3. Workshops: 1. Challenges in writing a research proposal.2. How to present your data.
2011 Infectious Diseases in Africa: Measurement of Immune Responses&
4th African Flow Cytometry Workshop
University of Cape Town, Cape Town 11-13 November 2011
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Topics of the Workshop: TBD
1. Instrument optimization for BD Calibur and LSR II
2. Panel Optimization
3. Assay standardization
4. Software training for DIVA, FlowJo, PESTLE, and SPICE
20011 Infectious Diseases in Africa: Measurement of Immune Responses&
4th African Flow Cytometry Workshop
University of Cape Town, Cape Town 11-13 November 2011
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
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.
2011 Infectious Diseases in Africa: Measurement of Immune Responses&
4th African Flow Cytometry Workshop
University of Cape Town, Cape Town 11-13 November 2011
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Way forward:1. To mentor young scientists
2. Create a network of young scientists3. To implement education of clinical staff:
open clinical/research laboratory to medical student
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Medical Education Partnership Initiative(MEPI)
• Goal to sustainably enhance and support medical education through partnerships between African and US schools of medicine
• Jointly sponsored by NIH and PEPFAR• Eleven awards announced October 2010• Kilimanjaro Christian Medical College-Duke University
School of Medicine awarded $10,000,000 over 5 years (Prof. Moshi and Dr. Bartlett P.I.)
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
SCHARPSteve Self Natalie Hawkins
MANAGEMENT COREKelly SoderbergTom DennyLiz PetzoldBart Haynes (Duke, US)
T CELL COREMichael LiuNilu GoonetillekeEmma TurnbullStephen MooreRachel TannerKati DigleriaTim RostronMcM CHAVI LABPersephone BorrowAndrew McMichael (Oxford, UK)
Christine MarkingRalph PickingKent Weinhold (Duke, US)
Mandla Mlotshwa Catherine RiouClive Gray (NICD, S.Africa)
The Patients.
CLINICAL COREMike Cohen (UNC, US)Marybeth McCauley (FHI, US)Joe Eron (UNC)Charles Hicks (Duke)Cindy Gay (UNC)Kara McGhee (Duke)Ian Williams (St Mary ,UK)Perry Pellegrino (St Mary, UK) VIRUS CORE
Bette Korber (LANL, US)
Jesus SalazarMaria Gonzalez-Salazar Brandon KeeleBeatrice Hahn George Shaw (UAB, US)
Jennifer Kirchner Chunlai JiangFeng Gao (Duke, US)
Florette Treurnicht Carolyn Williamson (UCT, S. Africa)
CHAVI SUPPORT TEAMS
CHAVI REPOSITORIESFHIDuke Management team
U. of PennsylvaniaMichael Betts
Vaccine Research Center/NIHMario Roederer
Acknowledgements
Presented at the 5th INTEREST workshop – 10 – 13 May 2010, Dar-es-Salaam, Tanzania
Co-organizer of IDA and Workshop
Clive Gray (NICD, South Africa)
The Senior and Junior Faculty and the Participants.
ACKNOWLEDGMENTS
Sponsored by: African AIDS Vaccine Initiative; National Institute of Health and Office For AIDS Research.
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.
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