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

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

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?

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

Features of 8-color staining panel used for HVTN trials

Marker Purpose

Violet Viability Dye Excludes Dead Cells

Defines Lineage

IFN-IL-2TNF-IL-4

Cytokine Response

CD3CD4CD8

8-Color Staining Profile for PBMC Stimulated with SEB

Example of Co-Expression of IFN- and IL-2 for SEB Stimulation

2.8% 1.7%

0.8%

1.2% 2%

5.8%

CD4+ T cells CD8+ T cells

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)

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

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

Example from recent HVTN trial

Env pool 1

CD4 CD8

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

Env pool 2

CD4 CD8IF

N-

on

lyIL

-2 o

nly

IFN

-+IL

-2+

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

Env pool 3

CD4 CD8

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

Gag pool 1

CD4 CD8

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

Gag pool 2

CD4 CD8

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

IFN

- o

nly

IL-2

onl

yIF

N-

+IL

-2+

AndPol pool 1Pol pool 2Pol pool 3

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.

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

PositivityEtc.

Export in format appropriate for

statistical analysis

Each experiment (“run”) includes multiple FCS files

Create new project

Organization of data files on server

Multiple runs

Multiple FCS files for each run

Upload FCS data files

Create Analysis Script

Script separated into compensation and analysis

Compensation editorKeyword in FCS file identifies comp samples

FlowJo template identifies positive cells

Upload FJ workspace

FlowJo templates uploaded for compensation and analysis

Analysis completed

List of completed analyses by run

View All Analyses and Filter

Can query to view all analyses for one or more runs

Filtering data to alter view

Filtered and sorted analysesSorted by sample and by peptide pool

View graphs - filtered for SEB

Quality control query - compensation samples

Acceptance based on minimum frequency of cells in gate

Link data from sample tableSample information from Excel

table is uploaded

Linking columns must be defined

Choose fields to join

Keywords in FCS file and in sample table must be defined to uniquely identify sample

Keywords in FCS fileKeywords in sample table

Quality control query - SOP-defined acceptance critera

Sample failed due to high background

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

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

Acknowledgements

•HVTN Laboratory Program

•SCHARP

•LabKey