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

Transcription

1 The Use of Flow Cytometry in Clinical and Basic HIV1 1 Research 4th INTEREST Workshop Maputo, Mozambique 2528 June 21 Guido Ferrari, MD gflmp@duke.edu Duke University Medical Center Center for AIDS Research (CFAR) International AIDS Training Research Program (IATRP) Duke Global Health Institute (DGHI) 2528 May 21, Maputo Mozambique

2 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 May 21, Maputo Mozambique

3 Basic Aspect of a Flow Cytometer Analog Digital 2528 May 21, Maputo Mozambique

4 Flow Cytometry: : A Quantitative Assay Platform Frequency of the Cellular Subset 1 Level of Expression 2528 May 21, Maputo Mozambique

5 The Achilles Heel of Flow Cytometry: Signal Spillover Non Compensated Parameter 1 Compensated Parameter May 21, Maputo Mozambique

6 What Can We Learn by Flow? Frequency of Cell Populations and their Functions 1. 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 Cells By using fluorescent compounds CFSE for lymphoproliferation assay (LPA) Intracellular Expression of Proteins or Infectious Agents 1. GFP expressed cloned in cell lines and infectious agents 2. Activated peptides (apoptotic mechanisms and killing ability) 2528 May 21, Maputo Mozambique

7 Flow Cytometry Applications in HIV1 1 Infection Clinical Applications Research Applications 2528 May 21, Maputo Mozambique

8 Clinical Application: Patient Management 1 Assay for CD4 Count and Rx Adherence CD4 Count: Clinical Stage CD8 Activation Virus Load Virus Load Rx Adherence CD8 Activation Rx Adherence Glencross et al. CD8/CD38 activation yields important clinical information of effective antiretroviral therapy. Cytometry (28) vol. 74B (S1) pp. S131S May 21, Maputo Mozambique

9 Please see Posters P_26: Coetzee L. et al.: Monitoring CD38 activation in HIV1 infected pts on Rx is a potentially costeffective alternative to repeated VL testing P_27: Pillay K. et al.: Extended window period for CD38 activation marker testing to 48 hours post venesection 2528 May 21, Maputo Mozambique

10 Flow Cytometry Applications in HIV1 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 Agspecific and nonagspecific Cells May 21, Maputo Mozambique

11 Detection of 8 parameters to identify all possibility for 4 combined parameters: 4color Cytometer = 25 tubes = 25 million cells = 25 ml of blood 8color Cytometer = 1 tube = 1 million cell = 1 ml of blood 488nm Blue Laser (2mW, Solid State) Duke LSRII Rm 23 FITC CFSE GFP Calcein 635nm Red Laser (25mW, Diode) Duke LSRII Alexa 68 Alexa 7 515/2 55L P 71/5 685L P 532nm Green Laser (15mW, Solid State) PE Gre en 575/ L P SS C PE Cy5 64L P 66/ May 21, Maputo Mozambique 685L P 71/ 4 PE Blu e 71/5 74L P 61/2 6LP 69L P 78/4 PE Cy7 PerCP Cy5.5 Duke LSRII APC Alexa 647 PerCPCy5.5 PE Cy5.5 PETR Alexa 61PE Am Cya n 45nm Violet Laser QD ot 565 (5mW, Diode) 515/ 2 56/ LP 55 LP 65/4 QD ot 65 66/ 2 595L P QDo t /4 63L P 74L P 57 LP 67L P 78/ 6 75/7 585/4 2 QD ot 75 56/ LP Duke LSR II 45/ 5 QDot 585 QDot 545 APCCy7 APCHL75 Alexa 75 Qdot 545 PacOrange Cascade Blue vamine PacBlue

12 1. Stimulate ICS Methodology 2. Viability&Surface Staining 3. Permeabilize 4. Stain Brefeldin A EDTA 6 h Wash Wash Wash cytokine lymphocyte erythrocyte A g Ag presenting cell T cell CD8 PE IFN FITC Brefeldin A IFN CD3 APC 5. Acquisition 6. Analysis Horton et al. Optimization and validation of an 8color intracellular cytokine staining (ICS) assay to quantify antigenspecific T cells induced by vaccination. Journal of Immunological Methods (27) vol. 323 (1) pp May 21, Maputo Mozambique

13 Gating Strategy for the ICS Assay Analysis I. Live Lymphocytes Subsets FSCH 25K 2K 15K 1K Singlets 77.9 Viability Live CD3 SSCA 25K 2K 15K 1K Lymphocytes 97.7 CD K K 1 2 5K 1K 15K 2K 25K FSCA CD CD3 CD8 Memory 5K 1K 15K 2K 25K FSCA CD Naive CD CD57 51 CD CD CD Memory CD45RO CD45R CD45R CD45R memory CD8 Functions CD17a 1 3 IFN 1 3 IL2 IL2 1 3 MIP1 1 3 CD17a IFN TNF TNF TNF TNF 2528 May 21, Maputo Mozambique

14 Boolean Logic in Flow Cytometry The Boolean logic in Flow Cytometry allows to identify populations that fell in different gates according to the And, Or, or Not principles. Subset 1 Not 2 Subset 1 And May 21, Maputo Mozambique

15 Multiparameter ICS to Identify Functional CD8 Memory Subsets memory CD8 Functions CD17a IFN IL2 IL MIP CD17a IFN TNF TNF TNF TNF II. III. Functional Family Pies CD17 IFN IL2 MIP1 TNF CD17a IFN IL2 MIP1 TNF Boolean Analysis CD17 or IFN or IL2 or MIP1 or TNF Combined Analysis = Boolean Analysis Total Functional CD8 Response by Memory Phenotype CD CM EM CD TE CD45RO May 21, Maputo Mozambique

16 Uniform Expansion of Activation markers in ED, LD and Effector antigenspecific CD4 Tcells 2528 May 21, Maputo Mozambique Maenetje P, G, Gray CM, et al. J Immunol. 21;184(9):

17 Flow Cytometers in Africa by just 1 company Flow Cytometry does not have to be highly complex and can be implemented in researchlimited setting if focused and aimed to study any of the parameters thus far presented May 21, Maputo Mozambique

18 3 Participants We received 11 applications from the countries indicated on the map. Twentyfour (24) applicants were selected for the symposia and 12 more for the African Flow Cytometry Workshops that followed the Symposium May 21, Maputo Mozambique

19 Organizing Committee * Scientific Committee and Faculty * * * * ** * * * * * * 2528 May 21, Maputo Mozambique

20 29 Infectious Diseases in Africa: Measurement of Immune Responses ses & 3rd African Flow Cytometry Workshop NICD, Johannesburg November May 21, Maputo Mozambique

21 29 Infectious Diseases in Africa: Measurement of Immune Responses Visit: Topics of the Symposium: 1. Faculty Seminars on Adaptive and Innate Immune Responses to HIV, Malaria, and TB; Presentations by Young Investigators on HIV, Malaria, and TB; 3. Workshops on writing research proposal May 21, Maputo Mozambique

22 29 Infectious Diseases in Africa: Measurement of Immune Responses & 3rd African Flow Cytometry Workshop NICD, Johannesburg November 29 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 2528 May 21, Maputo Mozambique

23 Infectious Diseases in Africa: Measurement of Immune Responses & 3rd African Flow Cytometry Workshop NICD, Johannesburg November 29 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 23 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 May 21, Maputo Mozambique

24 ACKNOWLEDGMENTS Coorganizer of IDA and Workshop Clive Gray (NICD, South Africa) The Members of the Organizing and Scientific Committees. The Faculty and the Participants. 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 May 21, Maputo Mozambique