Idea Transcript
HIGH DIMENSIONAL FLOW CYTOMETRY FOR COMPREHENSIVE IMMUNE MONITORING IN CLINICAL TRIALS
Dominic Gagnon, Yoav Peretz, Marylène For8n, Claire Landry, and David Favre
ImmuneCarta Services, 2901 Rachel Est, Suite 22, Montréal, QC, Canada, H1W 4A4
Introduc8on
Precision, Stability and Reproducibility
Study Cases of Immune Monitoring
ImmuneCarta Services is a leading provider of services for preclinical and clinical studies related to immunology. Over the past 7 years, we have developed a broad bayery of innova8ve assays to characterize cell popula8ons and immune responses in the sepng of infec8ous diseases, cancer, vaccine trials and immune-‐based therapies. Based in Montréal, ImmuneCarta Services is specialized in advanced mul8parametric flow-‐based assays performed according to GLP regula8ons, GCLP guidelines governed by Quality Management Systems and standard opera8ng procedures. ImmuneCarta exper8se includes the assessment of phenotypic and func8onal markers, the characteriza8on of cell subset lineages, ac8va8on states, and signaling molecules, as well as the quan8ta8ve analysis of vaccine-‐, pathogen-‐ or drug-‐specific responses based on an8body signatures, cytokine/ chemokine profiles, and signaling pathways. We describe here our experience as a contract research organiza8on providing services to the biopharmaceu8cal industry, in the execu8on of high dimensional flow cytometry analysis of subjects enrolled in Phase I/II clinical trials.
Flow cytometry is based on fluorescence, fluidic and op8cal tools with the help of signal and image computer treatment. ImmuneCarta Services uses 3-‐ and 4-‐laser LSR II Becton Dickinson instruments. These instruments are high performance systems allowing simultaneous analysis of up to 18 colors using automated sampler in 96-‐well plate format. High dimensional flow cytometry requires sensi8ve and precise methods with op8mal stability and reproducibility of the signal. For customized an8body panels, qualifica8on or valida8on steps are necessary to address specificity, precision, accuracy, lower and upper limits and range of detec8on, stability and reproducibility of the analysis. The precision and accuracy of flow cytometry experiments also depends on stable applica8on sepngs (CS&T beads) as well as internal and/or external quality control (QC) samples.
Since 2004, ImmuneCarta has applied a broad array of innova8ve assays for the biopharmaceu8cal industry and government ins8tu8ons to characterize the immune profiling of adap8ve and innate immunity and the potency of immune-‐related drugs or vaccines in exploratory and Phase I and Phase II clinical trials. ImmuneCarta Services recently formed a strategic alliance with Caprion Proteome Inc., the leading company in proteomics and biomarker discovery (www.caprion.com) in order to integrate single-‐ cell mul8parametric flow cytometry analysis with soluble markers, serological measurements and other large datasets including genomics and proteomics.
0 Beads
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102
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103 PerCP-A
Beads
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Over the past 7 years, we have implemented assays using high throughput analy8cal methods with 10 to 18-‐flow cytometry parameters on fresh and cryopreserved human peripheral blood samples. Overall, immune monitoring of clinical trials involve study planning, assay valida8on, specimen handling, assay execu8on, monitoring, repor8ng, and quality review performed as per applicable GLP regula8ons and GCLP guidelines governed by quality systems and standard opera8ng procedures.
• Innova8on and development program • Design of immune monitoring strategy and workplan • Ph.D.-‐level Principal Scien8sts assigned to each study
Standard and Customized Immune Monitoring Assays
• Involved in study set-‐up, lab manual wri8ng, and sample management • On-‐site training of clinical sites for maximum sample viability and recovery
Study Progress and Final Report to Sponsor • Conference calls, interim and final scien8fic reports • Quality Assurance Statement • Ac8ve par8cipa8on in scien8fic posters and publica8ons
• Assay development, qualifica8on, and valida8on; SOP & worksheets • Data integrity, high quality and high throughput processing and analysis • Documenta8on, sample tracking and control, GLP/GCLP training
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Count/uL
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Exported as “Experiment” from Diva database to Raw Data Server
Assay
Acquisi8on Sta8on
• Phenotyping • Func8onal profiling • Enumera8on • ICS • Prolifera8on • PhosFlow • Cyotkine bead arrays
Raw Data Server
• BD Diva/ LSR II • Daily and monthly QC • Acquisi8on template applied • Applica8on sepngs used • Compensa8on and staining references
K
Analysis Sta8on
• Users can only create or read files • Regular backup
Batch layout PDF, and data table saved in Derived Data Server
.jo, .csv, and .PDF from FlowJo saved in Derived Data Server
.fcs files read from Raw Data Server on FlowJo template
Derived Data Server
QC Sta8on
• Delete/modify restricted • Regular backup
• iMac analysis sta8on • Import FlowJo assay template • Exploratory analysis can also be done with SPICE/PRISM
QA if needed
• Another analyst on another iMac analysis sta8on • Verify ga8ng and documenta8on • Confirm data report across all files
Final data and report exported in Study Server
Client Report • Presenta8ons • Data Table • PDF
0.0154
CD4
DP
Lymph
29.2
70.8
CD3+
: CD4
SSC-A
: CD19
: CD45
: CD3
: CD19
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0.0614
0.157
0.0554
3.07e-3
0.734
0.0294
CD3B Cells
CD8+ T cells
NK
0.672
DN
CD8 : CD8
0.13
IFNg
1.68e-3
0.354
CD107a
TNFa
IL-2
IL-4
IL-17
Performed at ImmuneCarta Services
Other soluble markers
PhosFlow
DNA analysis (SNP/telomeres)
RNA/mRNA analysis
Figure 3: Example of sample management for assays performed at ImmuneCarta Services and at collaborator sites. In this vaccine clinical trial on 200 subjects, ImmuneCarta provided mul8ple services including study design, central lab ac8vi8es, assays and integrated analy8cal report. In this example, fresh blood samples from each subject and each 8me point were collected at a CRO site nearby and immediately processed for analysis at ImmuneCarta or stored at ImmuneCarta for analysis at collaborator sites, e.g. genomics, gene8c, other soluble markers or PhosFlow.
wk8
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90 mL
90 mL
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90 mL
ImmuneCarta DC TARGET PRODUCTION
ICS
CFSE
POST-‐THERAPY IM SAMPLES
CFSE
ICS and CFSE
CFSE
CFSE
High Dimensionality Analysis of Hyporesponsiveness to Protein Subunit Vaccines in the Elderly -- Introduction to Study MK0000-131 Carayannopoulos LN1,, Railkar RA1, Favre D2, Landry C2, Schaeffer AK1, Wiener MC1, Chastain M1, Loboda A1, Lukac S1, Duguay D3, Audet D3, St-Maurice F3, Kaslow DC1, Beals CR1, Sekaly RP2,4 1Merck Research Laboratories, USA | 2National Immune Monitoring Laboratory – Genome Quebec, Canada | 3Anapharm-Pharmanet Quebec, Canada | 4VGTI-Florida
Cytometric Vaccine Response Examples Plasmacyte Counts before (V2) and after (V3) immunization (left) All Plasmacytes (right) IgG+ Plasmacytes
Figure 8: Reproducibility and %CV from external QC samples: results obtained with BD MulN-‐check CD4 low controls in 14 experiments with lot # 40L in April 2010 and 17 experiments with lot # 50L in May 2010. The first graph represents the enumera8on of lymphocytes (CD45), T cell popula8ons (CD3, CD4, and CD8), B cells, and NK cells. The second graph represents the enumera8on of the same popula8ons. Coefficient of varia8on are indicated in blue.
Boolean Gating (total CD4+ and CD8+ T cells):
Beads
Performed at collaborator sites
wk0
ART: an8-‐retroviral treatment ARTI: ART interrup8on
Populations
High throughput analysis of high dimensional flow cytometry data requires advanced soWware and methods. Data analysis performed at ImmuneCarta Services relies on flow data acquisi8on using DiVa soWware (BD Biosciences), and data analysis with FlowJo (Treestar Inc.), Excel (Windows), PESTLE/SPICE (NIH), Cluster (Open source, Stanford), Java TreeView (Open source), Prism (GraphPad SoWware) and/or other specialized soWware for sta8s8cal analysis and systems biology. All data are acquired and analyzed in compliance with 21 CFR Part11 to ensure quality and integrity of the raw data and its analysis. Pre-‐defined FlowJo templates are qualified or validated (GLP study) prior to being used throughout studies and require minimal ga8ng adjustments that are documented accordingly.
Paxgene tube storage
Flow cytometry B cell panel (11 colors)
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NK Populations
CD4+ T cells
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Flow cytometry T cell panel (12 colors) Innate panel (12 colors)
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ImmuKnow Assay
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Figure 9: Flow of work for data analysis. Sample processing, assays and data acquisi8on are based on pre-‐approved worksheets related to study requirements. Samples are acquired on BD LSR II cytometers based on pre-‐defined sepngs and templates. The raw FACS data are exported directly to the Raw Data server and used on FlowJo analysis template on the Derived Data server. Once analysis has been completed and documented, a full QC of data analysis is performed. Final client data and report are generated, while the whole process is audited by the Quality Assurance (GLP studies). Ficoll
Follow-‐up & Safety
Figure 14: Study protocol and immune monitoring of the AGS-‐004 pilot study to invesNgate the safety and immunologic acNvity of an autologous HIV immunotherapeuNc agent. (NCT00381212) ICS and CFSE prolifera8on assays were performed as indicated in the study protocol (leW). The frequencies of the HIV(GNRV)-‐specific CD8 and CD4 T cell responses and their func8onal profiles (CD107a, IFNγ and IL-‐2) (right) are compared between baseline and visit 8 aWer vaccine administra8on. Pie charts represent the rela8ve distribu8on of the func8onal subsets within the total CD8 and CD4 T cell pools. Sta8s8cal significant differences (P < 0.05) between pre (V2)-‐ and post (V8)-‐vaccine 8me points are indicated by Wilcoxon-‐Rank and a Student’s t-‐test (# and +, respec8vely). The 90th and 75th percen8le threshold applied to background subtracted CD8 and CD4 T cell responses were 0.01% and 0.005%, respec8vely.
CD4 Low control, lot # 50L
1500
C D
0
Figure 2: Overview of immune monitoring services. ImmuneCarta Services include all steps from strategic planning to final reports to clients. It oWen involves study set-‐up, lab manual wri8ng and sample management which are cri8cal steps for cell-‐based analysis as flow cytometry assays. Customized assays as per client needs require assay development and valida8on prior to immune monitoring of clinical samples. All steps include an ac8ve follow-‐up with the sponsor, are performed using proprietary SOPs and worksheets and are documented as per applicable GLP regula8ons.
Serum aliquoNng/ storage
Booster Phase
Vaccine ARTI
Titer DistribuNons of Vaccinated Subjects
Figure 7: Reproducibility and %CV from internal QC samples (frozen PBMC, L747) on 11-‐color T, B, and Innate cell phenotype panels. Results from 32 experiments are shown over one year (July 2010 to September 2011). The first graph represents the enumera8on of T cell popula8ons (CD3, CD4, and CD8). The second graph represents the enumera8on of B cells, the monocytes, and sub-‐popula8ons of dendri8c cells. The third graph represents the enumera8on of natural killer (NK) sub-‐popula8ons. Coefficient of varia8on are indicated in blue.
Immune Monitoring
Pro-‐ac8ve Rela8onship with Sponsors, CRO, and Clinical Sites
1200 1600 2000
50
10
100
T Cells Populations
Strategic Planning and Communica8on
800
100
20
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150
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400
Immune Monitoring
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K
sjTREC
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40
ht
Cells, 8ssue
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T cell receptor excision circles (TREC)
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Specific mRNA quan8fica8on
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6+
mRNA expression (real-‐Nme PCR)
Cells, 8ssue
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wk24
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-1
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w20
6000
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Interleukins, sICAM-‐1, sICAM-‐3, sVCAM-‐1, sPECAM-‐1, sE-‐Selec8n, sP-‐ Selec8n, G-‐CSF, IL-‐8, MCP-‐1, MIG, MIP-‐1α, MIP-‐1β, others
1000
56
Serum, plasma, cell culture supernatant
800
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Cytokines/Chemokines/Adhesion Molecules/Growth Factors
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V3
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1200
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Tetanus, Diphtheria, Hepa88s B, Cholera toxin B, CMV, others
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Serum, plasma
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800
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Count / uL
AnNbody Titers
1500
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✓
10000
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CD107a, Granzyme, Perforin, CD63, others
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T, NK, NKT cells and subsets, Basophils
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Cytotoxicity/DegranulaNon
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IFNγ and/or IL-‐2, TNFα, IgG, IgM
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CD8+/CD4+ T cells, B cells
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ELISPOT
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CD3 CD4 CD8 DP DN B cells HLA-DR+ B cells HLA-DRMonocytes pDC mDC1 mDC2 All NK NK 56-16+ NK 56bright16NK 56low16NK 56low16+ NK 56bright16low
R2 = 0.9954
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NK L747
PD C
✓
• •
• • • • • • • • • • • • • •
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B, mono, and DC L747
T Cells L747
o
CFSE, Ki67, BrDU
• • • • • • • • • • • •
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w12
Vaccine ART
Client PRODUCT PRODUCTION
CD4 Low control, lot # 40L
on
T cells and subsets
• • • • • • • • • • • •
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Akt, Btk, Elk, EGF-‐R, Lck, LAT, Zap70, Syk, MEK1, NFκB, PKC, PLC-‐γ1, PLC-‐ γ2, p38MAPK, ERKk1/2 Src, STAT1 to STAT-‐6
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lls
T, B and Innate Cells, Tumor cells
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Annexin, caspase 3, CD95, PARP, TUNEL, Live/Dead, 7-‐AAD
Count / uL
T, B and Innate cells
ce lls
Gene Expression & DNA Analysis
Apoptosis/Necrosis
• • • • • • • • •
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0
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Cell counts (cells/µL)
Figure 6: Reproducibility of high dimensional flow cytometry. Phenotyping of T, B, and innate cells using 11-‐12 color an8body panels was assessed independently on two blood samples from 20 subjects at Visit 2 (V2 and V2-‐redo). Distribu8on of popula8ons and cell counts are highly correlated with R2 > 0.99.
B
Serology & Soluble Markers
✓
IL-‐2, TNFα, IFNγ, IL-‐4, IL-‐17, IL-‐22, IL-‐10, TGFβ, IL-‐9, IL-‐21, Mip1β, others
•
R2 = 0.9955
50
w8
Samples
2000
V2
D N
FuncNonal Profiling
T, B, NK, NKT, Dendri8c cells, Monocytes
• • • • •
100
DP
Intracellular Cytokine/Chemokine Staining
✓
FoxP3, T-‐bet, GATA-‐3, RORγt, BCL-‐6
80
ILD (CD8) LD (CD8) Naive (CD4) CM (CD4) EM (CD4) TEM1 (CD4) TEM2 (CD4) ILD (CD4) LD (CD4) Mem CD45RA- (CD8) Mem CD45RA- (CD4)
V2
8
Treg, Th1, Th2, Th17, T~
60
D
TranscripNonal Factors
✓
CCR4, CCR5, CCR6, CCR7, CCR9, CXCR3, α4β7 integrins, others
40
C
T cells, B and Innate cells
20
4
Homing Receptors/Co-‐receptors
• • • •
0
D
✓
0
C
HLA-‐DR, CD38, ICOS, OX40, 4-‐1BB, Ki67, CD40, CD95, PD-‐1, CD57, CD83, CD80, CD86, CD160, Lag-‐3, 2B4, CTLA-‐4, Tim-‐3
V2-Redo
T, B and Innate cells
3
AcNvaNon/InhibiNon/ExhausNon/ Immune Senescence
40
D
✓
60
20
C
An8gen-‐specific, HLA-‐restricted TCR (using Tetramer/Pentamer/Dexamer)
Chronic Inflamma8on
Tetramer-‐posi8ve an8gen-‐specific CD8+/CD4+ T cells
Immune Senescence
AnNgenic specificity
• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
CD28- (CD8) CD4 (CD3) CD8 (CD3) PD-1 (CD4) PD-1 (CD8) CD57 (CD4) CD57 (CD8) Naive (CD8) CM (CD8) EM (CD8) TEM1 (CD8) TEM2 (CD8)
w4
Innate cell Counts Switched IgG Switched IgA Unswitched IgM MZ-like Immature B cell Naive IgM-IgD+ Naive IgM+IgD+ Naive IgM+IgDMemory IgA+ Memory IgG+ Memory IgM+IgD+ Memory IgM+IgDMemory IgM-IgD+ Plasma B cells Plasma IgA+ Plasma IgD+ Plasma IgG+ Plasma IgM+
% Parent
60
R2 = 0.9956
80
Count / uL
Immune Phenotyping
Transplanta8on
•
Autoimmunity
✓
Cardiology
CD45RA/RO, CD27, CCR7, CD28, CD62L/CD27, IgD/M, CD20, CD38,CD10,IgA/G/E/CD94/NKG2A, CD7, KIR2DL/DS/CD11c,/CD123, BDCA-‐2/3/4, /FcγRs, IgE, CCR3/CD16, CD64, FcγRs
Oncology
T, B, NK and NKT cells, Dendri8c cells, Basophils, Monocytes
Allergy
Cell Subsets DifferenNaNon
Infec8ous diseases
✓
Main therapeuNc area Immunotoxicology
T, B, NK and NKT cells, Dendri8c cells, Basophils, Monocytes
Epitope Mapping
Cell Lineage
CD3, CD4, CD8/CD19, HLA-‐DR/CD16, CD56, Lin-‐/Vα24, αGalCerCD1d tetramer, CD3/CD11c, CD123, Lin-‐/CD123, Lin-‐/CD14
Vaccine
Immune Monitoring
DNA, Cytokera8n, CD45
Biologics/Biosimilars
Circula8ng Tumor Cells
Biomarkers
Tumor Cells
✓
CD34, CD45, others
Drug development
✓
Circula8ng CD34+
Molecular biology
CD3, CD4, CD8, CD16/56, CD19, CD14, CD11c, CD45, CD123, others
HematopoieNc stem cells
Field of applicaNon
Chemiluminescence
Cytokine bead array
Cell EnumeraNon
Markers
ELISA/ELISPOT
Flow Cytometry
T, B and Innate cells
Assays
Customizable
Immune Cells
• • • •
Targeted Sample or Cell PopulaNon
% Parent
V2-redo
Table I: Overview of ImmuneCarta assays in different fields of applicaNon and therapeuNc areas.
w0
Pre-‐treatment Phase
Figure 5: Reproducibility of CD34+ absolute counts using reference QCs over Nme. Three levels of CD-‐ Chex CD34 reference controls were used (3, 35, and 124 CD34 cells/µL) and two BD Stem Cell Control Kit (12.1 and 35.9 CD34 cells/µL). Coefficient of varia8on for CD-‐Chex level 1, 2, and 3 are respec8vely 8.98%, 3.84%, and 1.98%. For BD Stem cell low and high reference controls, the CV are respec8vely 6.94% and 3.48%.
B cell Subsets
T cell Subsets
Vaccine Treatment
Visit/Week
C
Figure 1: Overview of ImmuneCarta flow cytometry and analyNcal processes (example of immune profiling of mucosal CD4+ T cells by intracellular cytokine detecNon)
Plamorm
105
Figure 4: Stability of the signal using applicaNon seangs coupled with CS&T. Compbeads were stained 45 8mes with either FITC, PE or PerCP (3 month survey) and were used to determine compensa8on for 81 experiments using cytometer # 1 and 31 experiments using cytometer # 2. Voltages were determined by the Applica8on Sepngs linked to the daily CS&T Performance check. First row: 11-‐color cytometer, second row, 18-‐color cytometer with yellow-‐green laser (see increased posi8ve PE signal without increasing the background).
Subj-‐2
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Figure 13: SchemaNc of the drug discovery process.
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11
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Phase IV / Market
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103 FITC-A
Phase III
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102
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Frequency)(%CD8))
Ungated
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Phase I
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LSR II # 1
80
% of Max
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% of Max
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% of Max
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StaNsNcal analysis and idenNficaNon of group differences in funcNonal responses
PerCP
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LSR II # 2
Single-‐cell analysis (% funcNonal response)
PE
100
% of Max
Flow Cytometry
FITC
100
% of Max
Sample
FSC/SSC
Figure 10: Example of a simple hierarchical gaNng for T, B and NK cell enumeraNon. Lymphocytes and BD TruCount Beads are gated based on SSC/CD45 expression. CD3-‐posi8ve and CD3-‐nega8ve popula8ons are defined. From CD3-‐ gate, natural killer (NK) and B cells are discriminated based on CD16/CD56+ (NK) and CD19+ (B cells). From CD3+ gate, CD4+ and CD8+ T cells are defined as well as double nega8ve and double posi8ve T cells.
Figure 11: Example of Boolean gaNng of 6 funcNonal markers expressed in CD4+, CD8+ or memory subsets and effector T cells by ICS aoer anNgen-‐ sNmulaNon of cryopreserved PBMCs. Posi8ve responses for each of the markers are defined from the template analysis (IFNγ, CD107a, TNFα, IL-‐2, IL-‐4, and IL-‐17). Boolean ga8ng is generated by FlowJo for all possible combina8ons (IFNγ+/-‐ and CD107a +/-‐ and TNFα+/-‐, and IL-‐2+/-‐ and IL-‐4+/-‐ and IL-‐17+/-‐ ), e.g. leading to 2n different gates. In this example, n = 6, genera8ng 64 gates for 8 popula8ons of interest per sample.
Figure 12: Boolean gaNng analysis of 5 markers in mucosal CD4+ T cells using SPICE analysis. CorrelaNon of specific funcNonal signatures with immune acNvaNon (Ki67+). Combina8on of 3 among 5 markers (IFNγ, IL-‐2, IL-‐17, MIP1β, and TNFα) are displayed as dots and their means, as grey histograms. Sta8s8cally significant differences between HIV viral controllers and non-‐controllers are shown by # (Wilcoxon-‐Rank) and + (Student’s t-‐test).
Figure 15: High dimensionality analysis of vaccine hyporesponse in healthy elderly subjects (NCT01119703) Titer distribu8on of vaccine responses to Tetanus, Diphtheria and Hepa88s B vaccines in elderly subjects (leW) and example of increase frequency of highly characterized B cell popula8ons one week (V3) aWer vaccine administra8on (V2) (middle). “Plasma B cells” are characterized as singlet/lymphocyte/CD19+/HLA-‐DR+/CD3-‐/CD27+/CD10-‐/CD20-‐ cells. “Plasma IgG+ B cells” are plasma B cells expressing IgG on the cell surface. An example of heatmap represen8ng unsupervised clustering of high dimensional flow cytometric Boolean datasets of T, B and innate immune phenotyping (y-‐axis) is shown on the same cohort (N=120 subjects, x-‐axis) (right). Such unsupervised clustering of large flow cytometric datasets allows further analysis of genomic datasets or other large datasets by quan8ta8ve regression analysis related to individual immune profiling, as described in Loke, Favre et al., Blood 2010.
Conclusions Flow cytometry is a unique way to address complex cellular immunological profiling for drug development and Phase I-‐III clinical trials in infec8ous diseases, cancer, vaccine, transplanta8on, autoimmune disorders and related immunomodula8on-‐based therapies. High dimensional mul8parametric single-‐cell analysis is not only aimed to define mul8ple markers of different cell popula8ons simultaneously -‐though helpful when clinical sample availability is limited-‐, it is also one of very few analy8cal plazorms that can address complex protein-‐based signatures (biomarkers, disease stage, etc.) and func8onal networks (mechanisms) from relevant and well-‐characterized primary human or animal cells at the single cell level. The immune monitoring of Phase I to Phase III clinical trials aims to design, perform and interpret immunological data that enable industry to move vaccines, immunotherapeu8cs and drug candidates through the regulatory process (FDA, EMEA, others). High dimensionality flow cytometric analysis also allows for the defini8on of immunological profiles that are disease and stage specific, enabling elimina8on of many unsuitable drug, vaccine or therapy candidates prior or at the 8me of “in-‐man” studies. This requires both a scien8fic exper8se in immunology, physiology and pathology as well as a clear understanding of technicali8es related to instruments, reagents and high dimensional data mining. As a service company for the pharmaceu8cal industry, ImmuneCarta regulatory process and standardized procedures are cri8cal to ensure data integrity and quality, especially when interpre8ng complex data sets to define disease stage, drug efficacy or toxicity. Overall, immune assays for diagnos8c, research or biomarker discovery may impact on all aspects and stages of immune system tes8ng, vaccine and immunotherapeu8c design and development as well as drug screening. They are enablers, permipng GO/ NO-‐GO decision-‐making, thus saving both 8me and money, enhancing safety and providing surrogate markers of clinical efficacy and/or mechanis8c insights.