1. Flow cytometry to evaluate vaccine-induced T cell responses: standardized analysis of large numbers of FCS files Stephen De Rosa, M.D. HVTN Laboratory Program, Fred Hutchinson Cancer Research Center and Department of Laboratory Medicine, University of Washington

2. Measurement of vaccine-induced T cell responses Intracellular Cytokine Staining (ICS) used to enumerate cytokine-producing T cells Responses are specific for vaccine inserts  In vitro stimulation with proteins or peptides encoded by vaccine Assay determines if vaccine is immunogenic  Number of vaccine recipients who respond  Magnitude of response in these individuals Results used as trial endpoint

3. Challenges As vaccine trials move from Phase I to Phase II and III, larger number of participants are included and larger numbers of assays are performed The flow-based ICS assay requires specialized analysis  Compensation  Sequential gating Manual analysis of each individual FCS file is not feasible Can the analysis be standardized and automated?

4. Standardized analysis Standardized assay procedures and standardized instrument set-up allow for standardized analysis Therefore, automated analysis is feasible LabKey has developed a semi-automated web-based system to analyze ICS data generated from clinical trials within the HIV-Vaccine Trials Network (HVTN)  Template gates are determined manually by examination of a few data sets  Template also determines which graphs and which statistics to generate  Automated analysis of all data sets within a trial are analyzed using the template

5. Features of 8-color staining panel used for HVTN trials Marker Purpose Violet Viability DyeExcludes Dead Cells Defines Lineage IFN-  IL-2 TNF-  IL-4 Cytokine Response CD3 CD4 CD8

6. 8-Color Staining Profile for PBMC Stimulated with SEB

7. Example of Co-Expression of IFN-  and IL-2 for SEB Stimulation 2.8%1.7% 0.8% 1.2%2% 5.8% CD4 + TcellsCD8 + Tcells

8. Data generated to measure T cell responses 8-color assay validated for IFN-  and IL-2 Three cytokine subsets for CD4 + and CD8 + T cells  IFN-  only, IL-2 only, IFN-  + IL-2 + Negative control (no antigen stimulation) Positive control (SEB = polyclonal T cell stimulation) Antigen-specific responses (to one or more peptide pools)

9. Qualitative determination of positive responses Statistical method based on antigen-specific response relative to negative control Applied to each cytokine subset Results in many different positivity calls for each PBMC sample

10. Example from recent HVTN trial Peptide pools used for in vitro stimulation Each protein requires multiple peptide pools to cover three proteins encoded in vaccine: Env, Gag, Pol  Env pools 1, 2 and 3  Gag pools 1 and 2  Pol pools 1, 2 and 3

11. Example from recent HVTN trial Env pool 1 CD4CD8 IFN-  only IL-2 only IFN-  + IL-2 + IFN-  only IL-2 only IFN-  + IL-2 + Env pool 2 CD4CD8 IFN-  only IL-2 only IFN-  + IL-2 + IFN-  only IL-2 only IFN-  + IL-2 + Env pool 3 CD4 CD8 IFN-  only IL-2 only IFN-  + IL-2 + IFN-  only IL-2 only IFN-  + IL-2 + Gag pool 1 CD4CD8 IFN-  only IL-2 only IFN-  + IL-2 + IFN-  only IL-2 only IFN-  + IL-2 + Gag pool 2 CD4CD8 IFN-  only IL-2 only IFN-  + IL-2 + IFN-  only IL-2 only IFN-  + IL-2 + And Pol pool 1 Pol pool 2 Pol pool 3

12. Positivity reported in multiple ways For any peptide pool, for any T cell, for any cytokine For each peptide pool, for any T cell, for any cytokine For any peptide pool, for CD4 or CD8 T cells, for any cytokine For each peptide pool, for CD4 or CD8 T cells, for any cytokine Etc.

13. Overview of automated analysis procedures Calculate compensation for each run Apply template gates to compensated FCS files Create FACS plots and determine frequencies of populations of interest Multiple views of data and graphs Queries for: Quality control Positivity Etc. Export in format appropriate for statistical analysis Each experiment (“run”) includes multiple FCS files

14. Create new project

15. Organization of data files on server Multiple runs Multiple FCS files for each run

16. Upload FCS data files

17. Create Analysis Script Script separated into compensation and analysis

18. Compensation editor Keyword in FCS file identifies comp samples FlowJo template identifies positive cells

19. Upload FJ workspace FlowJo templates uploaded for compensation and analysis

20. Analysis completed List of completed analyses by run

21. View All Analyses and Filter Can query to view all analyses for one or more runs Filtering data to alter view

22. Filtered and sorted analyses Sorted by sample and by peptide pool

23. View graphs - filtered for SEB

24. Quality control query - compensation samples Acceptance based on minimum frequency of cells in gate

25. Link data from sample table Sample information from Excel table is uploaded Linking columns must be defined

26. Choose fields to join Keywords in FCS file and in sample table must be defined to uniquely identify sample Keywords in FCS file Keywords in sample table

27. Quality control query - SOP-defined acceptance critera Sample failed due to high background

28. Clinical trial data System successfully used to process data from multiple trials Very few samples required use of non-standard gates Queries quickly identified samples that needed to be repeated Data output from system sent to SCHARP for statistical analysis

29. Future directions System is free and available under open source (cpas.fhcrc.org) The LabKey-developed flow system is in beta- testing and further development is in progress Future work includes: Easier-to-understand workflow Enhanced annotation with improved tracking Gate definition and editing in system Improved ability to use non-standard gates

30. Acknowledgements HVTN Laboratory Program SCHARP LabKey