1. “Measuring Antigen Specific T- cells using Surface and Intracellular Staining Polychromatic Flow Cytometry” 3 rd Annual CFAR Flow Cytometry Workshop 6-10 May, 2013 Janet Staats Flow Cytometry Core Facility Center for AIDS Research Duke University Medical Center E-mail: jotti@duke.edu

2. Part 1 of 3 Overview of PFC Assay Duke University Medical Center

3. IL-4IL-2 TNF  IFN  APC-T cell interactions Cytokine/Chemokine expression Rantes Apoptosis Proliferation/ Death Memory CD4 T Cell Response to Ag From H. Maecker Duke University Medical Center

4. CD4 + T cell cytokines CD8 + CTL APC MHC II CD4 CD8 cytokines Ag peptide MHC I T, B, or APC MHC I Whole protein Optimal peptide Duke University Medical Center From H. Maecker

5. Response to CMV pp65 Peptide Mix 0.19%2.03% pp65 proteinpeptide mixA2 peptide 1.14% CMV lysate 0.87% CD8 7.41%0.27%0.04%0.27% CD4 Duke University Medical Center From H. Maecker

6. Peptide Mixes 15 a.a. 11 a.a. CMV pp65: pool of 138 peptides HIV p55: pool of 120 peptides Duke University Medical Center

7. 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-  TNF  IL-2 Duke University Medical Center

8. Wash 5. Permeabilize Wash 6. IC Stain 7. Acquisition 8. Analysis Overview of 11-Color Assay 4. Lyse/Fix Brefeldin Monensin 3. Surface Stain 2. Stimulate Wash lymphocyte erythrocyte cytokine 6 hrs Amine CD14 CD3 CD4 CD8 CD45RO CD27 CD57 IFN  IL2 TNF 1. Thaw Rest CD107 6 h Costim SEB CMVpp65 Wash CD107 PE-Cy5 CD8 + CM Response 7+g+M+ g+M+ M+ MondayTuesdayWednesday Thursday - Friday Duke University Medical Center

9. FSC-W FSC-H 88.3 : CD8 Q705 : CD4 CY55PE 57.8 36.3 0.79 FSC-A SSC-A 99.3 : CD3 Amcyan : vAmine CD14PB CD19 PB 41.4 Gating Strategy for 11-Color Maturation/Function Panel: 1 of 3 CD4 PerCP-Cy5.5 SSC-A Exclusion (Violet H) FSC-H FSC-ACD3 AmCyanFSC-W CD8 Alexa700 UngatedSingletsCD3+ Exclusion- Scatter Basic Gates: CD4 + CD8 - CD8 + CD4 - CD4 + CD8 + - 3 total Duke University Medical Center

10. : CD27 CY5PE 43 54.1 2.58 0.33 : CD27 CY5PE 56.4 28.6 8.46 6.55 : CD57 Q545 0.12 1.07 55.9 42.9 : CD57 Q545 5.67 13.2 24.2 56.9 Gating Strategy for Sampson 11-Color Maturation/Function Panel: 2 of 3 : CD27 CY5PE 22 62.5 11.73.98 : CD57 Q545 3.98 22.9 51.721.5 CD57 FITC CD27 APC-Alexa750 CD45RO ECD N N N CM EM TE E E E Maturational Gates: CD4 + CD8 - CD8 + CD4 - CD4 + CD8 + CD45RO ECD Naive Central Memory Effector Memory Terminal Effector Effector Naive Central Memory Effector Memory Terminal Effector Effector Naive Central Memory Effector Memory Terminal Effector Effector - 5 per basic subset Duke University Medical Center

11. : CD107a AX680 2.59 CD107 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 = CD107 g = IFN-  2 = IL-2 T = TNF-  1.14 IL-2 TNF-  IFN-  0.31 4.19 Duke University Medical Center

12. 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 Roederer Immunotechnology Section VRC / NIAID / NIH Duke University Medical Center

13. Part 2 of 3 PFC Challenges Duke University Medical Center

14. 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!) Duke University Medical Center

15. Consistency across batches CD38 vs HLA-DR Staining on Ctrl 5L 28Feb08 5L CD8+ 04Marb08 5L CD8+ 11Mar08 5L CD8+ 06Mar08 5L CD8+ Duke University Medical Center

16. uncompensated compensation FSC/SSC settings PMT settings highlow Difficulties in doing Automated Analysis related to Instrument Settings CD4 CD3 IFN  CD69 CD4 SSC FSC SSC optimal Duke University Medical Center

17. 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!) Duke University Medical Center

18. Optimization using Spillover Assessments: Using Titration Files to Assess Spreading Error Violet G- CD3 AmCyan Red 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 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 2006 Duke University Medical Center

19. 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 CD3 AmCyan 9.8e-4 Unstained SSC 0.047 Blue B Unstained 66.3 4.58 0.13 CD57 FITC CD3 AmCyan 20.5 Duke University Medical Center

20. Is this positive??? CMV pp65 stimulated sample Maecker, et. al. Duke University Medical Center

21. Tandems Degrade! Ice Ice Dark Dark Fix Fix Controls Controls 6 hours 6 hours Maecker, et. al. Duke University Medical Center

22. Challenges… Instrument - optical configuration, optimization, standardization, and calibration Reagent - optimization and standardization Sample processing Staining protocols Data Analysis - compensation & gating Volume of data (death-by-excel!) Duke University Medical Center

23. 9-Color Activation/Maturation Using Cryo-preserved PBMC Duke University Medical Center

24. Batch Processing Error CD38 vs HLA-DR Staining on Ctrl 5L 28Feb08 5L CD8+ Lot 05262 04Marb08 5L CD8+ Lot 05262 11Mar08 5L CD8+ Lot 05262 06Mar08 5L CD8+ Lot 05262 26Feb08 5L CD8+ Lot 05262 Duke University Medical Center

25. Challenges… Instrument - optical configuration, optimization, standardization, and calibration Reagent - optimization and standardization Sample processing Staining protocols Data Analysis - compensation & gating Operator Volume of data (death-by-excel!) Duke University Medical Center

26. How would you gate? Markers:CD3CD4CD8IL-2+IFNg(FSC)(SSC) Duke University Medical Center

27. NCMEMTEE Pre-Vaccination 33% 21% 27% 2% 17% Post-Vaccination 8% 48% 25% 2% 17% Duke University Medical Center Reproducible analysis allows us to measure an expansion of CD4+ CM cells post vaccination with some degree of confidence

28. 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, 6:13http://www.biomedcentral.com/1471-2172/6/13 Duke University Medical Center

29. CIC ICS Gating Panel 110 labs participate d and there were 110 different approache s to gating

30. Before Backgate After Backgate IFNg Backgate CD3 AmCyan Exclusion 0.385.74 CD4 GatedCD8 Gated 5.23 0.27 IFNg PE-Cy7 CD4 PerCP-Cy5.5 CD8 APC-Cy7 Before Backgate After Backgate A B BACKGATING: purity & recovery Duke University Medical Center

31. Gating bias in proficiency panel results CD4 FITC IL2+IFN  PE UnstimCEFCMV pp65 0.02 % 0.01 % 0.16 % 0.03 % 0.02 % 0.17% 0.02 % 0.03 % 0.21 % Duke University Medical Center

32. We NEED better analysis tools!!! 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-  + IL-2 PE Expert Gating Manual Cluster Gating Automated Duke University Medical Center

33. 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 Range RCV? Duke University Medical Center

34. Challenges… Instrument - optical configuration, optimization, standardization, and calibration Reagent - optimization and standardization Sample processing Staining protocols Data Analysis - compensation & gating Operator Volume of data (death-by-excel!) Duke University Medical Center

35. Assay Complexity Duke University Medical Center

36. Endpoints for 11-Color Maturation/Function Panel DEATH BY EXCEL …….. Basic (3)Maturation (5)FunctionBoolean (16) CD4 + CD8 - CD4 + CD8 + CD4 - CD8 + Naïve Central Memory Effector Memory Effector Terminal Effector CD107 IFN-  IL-2 TNF-  Basic (3)Maturation (5)Boolean (16) XX 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/trial Duke University Medical Center

37. Data Annotation - for all 143,280 data points! Study ID Method Assay Name Batch # Operator Sample ID Visit ID Accession # % Viable (Flow) % Viable (Guava) Recovery CD4 count CD8 count Gate Name (Parameter Names) Tube Name File Name Error Code (1-11) Checking: X1 - for electronic data X3 - 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!!! Duke University Medical Center

38. Part 3 of 3 Why does this matter?? Why are you here??? Duke University Medical Center

39. 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 stimulation HIV negative donor Example CMVpp65 stimulation CMV positive donor % CD8+ CFSE low Laboratory Duke University Medical Center

40. History of Flow-based Proficiency/Standardization Efforts Duke University Medical Center

41. 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 Duke University Medical Center

42. DAIDS ICS Proficiency: Round 6, 26Jun09 (CMVpp65) CD4-CD8+CD4+CD8- IFN  + IL-2 PE CD3 APC-Cy7 Rep #1 Rep #2 Rep #3 Duke University Medical Center

43. Acknowledgements Duke CFAR Kent Weinhold Jennifer Enzor Twan Weaver Jianling Shi Cliburn Chan Patricia D’Souza (DAIDS) CFSE Standardization: Claire Laundry (NIML) EQAPOL Duke Tisch Brain Tumor Center Gary Archer Duane Mitchell John Sampson CHAVI VRC Steve Perfetto Laurie Lamoureaux Mario Roederer CVC Sylvia Janetski Duke DTRI Scottie Sparks (Roche)