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ProImmune Pentamer Day Pro5® MHC Pentamers and their Applications Dr. Helen Barnby-Porritt © Copyright ProImmune Limited 2011. All Rights Reserved Overview • Antigen-presentation / MHC function • Pro5® MHC Class I Pentamers • Primary Applications – Flow cytometry – Antigen-specific cell separation – Intracellular Cytokine Staining • CD1d tetramers © Copyright ProImmune Limited 2011. All Rights Reserved MHC Molecules Enable T Cells to Recognize and Eradicate Foreign Material in Tissues MHC molecules present intracellular material to CD8+ T cells APC peptides from virus particles, genes causing cancer, etc. MHC class I + peptide CD8+ T cells are activated by foreign, unrecognized peptides TCR co-stimulators Cytotoxic immune response © Copyright ProImmune Limited 2011. All Rights Reserved CD8+ T cell • Proliferation • Cytokine production • Cytotoxic effector proteins (Granzyme B, Perforin) Why use Pro5® MHC Pentamers? • Fluorescently-labeled soluble proteins that mimic surface MHC-peptide complexes • Bind specifically to the TCR • Sensitive detection of T cells reactive to a single MHCpeptide complex • Flexible-labeling technology • Most commonly cited commercial MHC multimer technology • Consistent and reproducible results • Rapid delivery (stock and custom synthesis) © Copyright ProImmune Limited 2011. All Rights Reserved Pro5® MHC Pentamers 5 MHC-peptide complexes Coiled-coil domain 5 Fluorescent tags © Copyright ProImmune Limited 2011. All Rights Reserved Primary Applications of Pro5® MHC Pentamers Flow cytometry Antigen-specific T cell isolation Intracellular Cytokine Staining © Copyright ProImmune Limited 2011. All Rights Reserved Flow Cytometry • Frequency determination of rare, antigen-specific T cell responses • Freshly isolated or frozen cells and T cell lines • Combine with antibody staining for activation markers or intracellular proteins such as cytokines © Copyright ProImmune Limited 2011. All Rights Reserved Labeled Pentamer Staining Protocol + 1 x 106 cells in 50ul per tube + 1 test MHC Pentamer (e.g. R-PE, APC or biotin) 10 minutes room temp. then wash anti-CD8 mAb (e.g. FITC) Fluorescentlabeled Streptavidin Pentamer 20 minutes on ice 2 x washes CD8 Analyze by flow cytometry © Copyright ProImmune Limited 2011. All Rights Reserved Unlabeled Pentamer Staining Protocol + + 10 minutes room temp. then wash 1 x 106 cells in 50ul per tube 1 test unlabeled MHC Pentamer Pro5 Fluorotag (e.g. R-PE) anti-CD8 mAb (e.g. FITC) Pentamer 20 minutes on ice 2 x washes CD8 Analyze by flow cytometry © Copyright ProImmune Limited 2011. All Rights Reserved Cells Stained for Flow Cytometry CD8 T cell receptor T cell R-PE-labeled Pentamer anti-CD8-FITC © Copyright ProImmune Limited 2011. All Rights Reserved Flow Cytometry - Sample Intake © Copyright ProImmune Limited 2011. All Rights Reserved Flow Cytometry - Optical Design PMT 5 PMT 4 Sample PMT 3 Flow cell Dichroic Filters Scatter Sensor PMT 2 PMT 1 Bandpass Filters © Copyright ProImmune Limited 2011. All Rights Reserved Laser Flow Cytometry Data SSC-H Pentamer R-PE CD8+ Pentamer+ (Antigen-specific) population of interest FSC-H CD8 FITC • Lymphocyte population is determined and gated upon • Plot of CD8 vs. Pentamer drawn based on this gate © Copyright ProImmune Limited 2011. All Rights Reserved Staining on Human Influenza-Specific Peripheral Blood Cells A*02:01 / GILGFVFTL (FluMP) Irrelevant control B*08:01 / RAKFKQLL (EBV BZLF-1) 0.0% 0.0% Pentamer 1.4% CD8 Human PBMCs 15,000 live events 10ul Pentamer CD8 clone LT8 © Copyright ProImmune Limited 2011. All Rights Reserved 0.0% A*02:01 Negative Control Pentamer A*02:01 / NLVPMVATV (CMV pp65) A*02:01 / Negative Pentamer 0.00% 0.05% A2 Neg Pentamer A2/CMV Pentamer 0.04% CD8 FITC © Copyright ProImmune Limited 2011. All Rights Reserved CD8 FITC 1.06% A*02:01 / ELAGIGILTV (Melan-A/MART-1) A*02:01 / ELAGIGILTV Stronen et al. (2009). Scand J. Immunol. 69: 319-328 CD8 APC A*02:01 / ELAGIGILTV staining immediately after isolation) A*02:01 / ELAGIGILTV staining after 12 days of co-culture with ELAGIGILTV peptide-pulsed DCs © Copyright ProImmune Limited 2011. All Rights Reserved Case Study: Validation of Trovax® epitopes using ProVE® Pentamers Harrop et al (2008) Cancer Immunol. Immunother. 57: 977-86 • Modified Vaccinia Ankara (MVA) expressing tumour antigen, 5T4 • Administration by intramuscular injection • Induces humoral and cellular immune response to 5T4 © Copyright ProImmune Limited 2011. All Rights Reserved Stage IV Colorectal Cancer – TroVax® + Chemotherapy Phase II Clinical Trial Design • Cycle of TroVax® injections • Accompanied by chemotherapy • Last TroVax® injections following end of chemotherapy Immune monitoring & 5T4 epitope discovery • Good 5T4 responses detected, e.g. by ELISPOT • Analysis of immune responses with PEPscreen® • MHC peptide binding assay • Validation with ProVE® MHC Pentamers © Copyright ProImmune Limited 2011. All Rights Reserved Staining on Cancer-Specific PBMCs Following Trovax® Vaccine Delivery Harrop et al (2008) Cancer Immunol. Immunother. 57: 977-86 Irrelevant Pentamer HLA-A2 5T4 Pentamer 0.18% 0.22% 5T4 Pentamer 0.00% Baseline CD8+ X+2wk X+14wk ELISPOT Analysis Baseline © Copyright ProImmune Limited 2011. All Rights Reserved X+2wk X+14wk Staining on Murine Cells Splenocytes from Ovalbumin peptide-immunized mice H-2Kb/SIINFEKL (Ovalbumin) 0.39% Pentamer 0.03% CD8 Immunized Murine splenocytes 50,000 live events 10ul Pentamer CD8 clone KT15 © Copyright ProImmune Limited 2011. All Rights Reserved Negative control H-2Kb/SSIEFARL (HSV-1 gp B) 0.01% 0.00% Staining with multiple Pentamers • Precious cell samples, e.g. derived from childhood diseases • Many different specificities to test • Library of MHC Pentamers − Use 3 different pre-labeled Pentamers − Screen with up to 6 unlabeled Pentamers © Copyright ProImmune Limited 2011. All Rights Reserved 0.00% 0.04% 0.02% CD8 FITC 0.00% 0.02% A2/CMV Bio + SA-PerCP B7/CMV Pentamer APC A2/flu Pentamer R-PE CD8 FITC 0.01% B7/CMV Pentamer APC 0.03% 0.27% 0.00% 0.02% A2/CMV Bio + SA-PerCP © Copyright ProImmune Limited 2011. All Rights Reserved 0.25% CD8 FITC A2/flu Pentamer R-PE 0.01% A2/CMV Bio + SA-PerCP A2/flu Pentamer R-PE Triple Staining on Human PBMCs 0.04% 0.00% 0.27% B7/CMV Pentamer APC Rapid Screening with a Pool of Unlabeled Pentamers Pentamer (a) Pool of 6 unlabeled Pentamers 0.11% 0.19% (b) A*01:01/Flu (CTELKLSDY) 0.07% 0.01% A*02:01/Flu (GILGFVFTL) 0.02% 0.06% A*24:02/CMV-1 (VYALPLKML) 0.01% 0.00% CD8 FITC A*24:02/CMV-2 (QYDPVAALF) Human PBMCs 100,000 live events 2ul Pentamer 8ul R-PE Fluorotag CD8 clone LT8 • 0.05% 0.00% B*07:02/CMV (TPRVTGGGAM) 0.06% 0.01% B*08:01/EBV (FLRGRAYGL) 0.02% 0.11% Pentamer pools that yield positive Pentamer staining can be dissected to resolve the antigen-specific populations © Copyright ProImmune Limited 2011. All Rights Reserved Phenotypic Characterization Stain for additional markers in conjunction with Pentamer staining to obtain as much information as possible from one batch of cells • Detailed Phenotype of the cell • Memory / activation status • Cytokine / Chemokine receptor expression • Homing characteristics © Copyright ProImmune Limited 2011. All Rights Reserved Phenotypic Characterization ProVE® Pentamer versus CD8 (naïve T cell marker) (memory T cell marker) 0.95% Pentamer CD8 0.11% 0.20% Pentamer 1.19% ProVE® Pentamer versus CD45RO Pentamer 0.04% ProVE® Pentamer versus CD45RA CD45RA APC Vaccinated Human PBMCs 500,000 live events 0.5ug ProVE™ Pentamer CD8 clone RPA-T8 CD45RA clone HI100 CD45RO clone UCHL-1 © Copyright ProImmune Limited 2011. All Rights Reserved CD45RO APC 1.00% Applications of Pro5® MHC Pentamers Flow cytometry Antigen-specific T cell isolation Intracellular Cytokine Staining © Copyright ProImmune Limited 2011. All Rights Reserved Antigen-Specific T Cell Isolation (1) Fluorescence-Activated Cell Sorting (FACS) (2) Magnetic Bead Separation © Copyright ProImmune Limited 2011. All Rights Reserved Fluorescence-Activated Cell Sorting (FACS) • Fluorescently labeled MHC Pentamers bind antigen-specific T cells • FACS instrument used to sort cells according to fluorescent properties • Used to isolate a pure population of live T cells (highly specific) © Copyright ProImmune Limited 2011. All Rights Reserved Principles of FACS Laser analysis point charge delay = 10 l break-off point Charged deflection plate l Population 2 Population 1 Waste © Copyright ProImmune Limited 2011. All Rights Reserved FACS data A*02:01/GLCTLVAML (EBV) Pentamer-specific cells sorted using a BD FACS Aria Pre-sort Post-sort 0.4% Pentamer 86.4% CD8 © Copyright ProImmune Limited 2011. All Rights Reserved Magnetic Bead Separation • Cells stained with Pentamer • Cells incubated with magnetic beads that recognize Pentamer • Antigen-specific cells isolated by magnetic separation • Two methods : − Column based − Tube based © Copyright ProImmune Limited 2011. All Rights Reserved Magnetic Bead Separation (Column-based) Wash T cell Place on column. Flow through collected as negative fraction R-PE labeled Pentamers are bound to T cells then detected by anti-R-PE microbeads Enriched cells labeled with anti-CD8 mAb (e.g. FITC) for analysis © Copyright ProImmune Limited 2011. All Rights Reserved Column removed from magnetic field and retained target cells flushed out as positively selected cells Column-based Magnetic Bead Enrichment MACS® anti-R-PE microbeads and single-step purification Pre-enrichment Post-enrichment 0.1% 0.6% 6.6% 51.4% B*07:02/TPRVTGGGAM (HCMV pp65) 0.2% 0.8% 4.3% 38.9% Pentamer B*08:01/RAKFKQLL (EBV BZLF-1) CD8 © Copyright ProImmune Limited 2011. All Rights Reserved Column-based Magnetic Bead Separation A*02:01/GLCTLVAML (EBV BMLF-1) Pentamer-specific cells separated with MACS® anti-R-PE microbeads in two steps Pre-sort 0.30% 8.63% Pentamer 0.08% Positive fraction after 1 round CD8 © Copyright ProImmune Limited 2011. All Rights Reserved 38.85% Positive fraction after 2 rounds 14.7% 79.08% Magnetic Bead Separation (Tube-based) Wash T cell Biotin-labeled Pentamers are bound to T cells then detected by Streptavidin microbeads Enriched cells labeled with anti-CD8 mAb (e.g. FITC) for analysis © Copyright ProImmune Limited 2011. All Rights Reserved Place on magnet. Target cells attracted to side of tube. Supernatant collected as negative fraction. Tube removed from magnetic field and remaining positivelyselected cells washed. Tube-based Magnetic Bead Separation B*08:01/RAKFKQLL (EBV BZLF-1) Pentamer-specific cells separated with LodeStars™ 2.7 Streptavidin beads (Varian Inc.) Pre-depletion Post-depletion B8/EBV Pentamer 1.53% CD8 97.4% of antigen-specific cells removed © Copyright ProImmune Limited 2011. All Rights Reserved 0.04% Column-based Magnetic Bead Enrichment in a life-saving treatment for PTLD Uhlin, M. et al. (2009). A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation. Cancer Immunol. Immunother. 59(3):473 The Patient • 18 year old woman with acute leukaemia. • Limited or no response to chemo and other drugs. • Received cord blood transplant (similar to bone marrow transplant). • Following transplant patient developed EBV linked Post-transplant lymphoproliferative disorder (PTLD): this is a disease caused by a common virus infection (EBV) and can occur after transplantation due to immune suppressants that have to be given after transplantation. • PTLD is like cancer and if not treated successfully is lethal like cancer. © Copyright ProImmune Limited 2011. All Rights Reserved Column-based Magnetic Bead Enrichment in a life-saving treatment for PTLD • Blood donated by mother; EBV-specific cells were stained with A*02:01 / GLCTLVAML or A*02:01 / CLGGLLTMV Pro5® MHC Pentamer • 600,000 cells were isolated with CliniMACS® and adoptively transferred to the patient from the mother • 189 days later, EBV lymphoma regression confirmed by CT scan Please note that ProImmune Products are sold for Research Use Only (RUO) © Copyright ProImmune Limited 2011. All Rights Reserved The Result • Donor-derived EBV-specific T cells expanded in vivo • Complete elimination of EBV virus from the patient • Patient currently alive 3 years after treatment and cured of PTLD Before treatment © Copyright ProImmune Limited 2011. All Rights Reserved After treatment The Result • Donor-derived EBV-specific T cells expanded in vivo • Complete elimination of EBV virus from the patient • Patient currently alive 3 years after treatment and cured of PTLD Before treatment © Copyright ProImmune Limited 2011. All Rights Reserved After treatment Applications of Pro5® MHC Pentamers Flow cytometry Antigen-specific T cell isolation Intracellular Cytokine Staining © Copyright ProImmune Limited 2011. All Rights Reserved MHC Pentamer & Intracellular Cytokine Staining (ICS) • Determine the frequency and effector function of antigen-specific T cells simultaneously • Co-staining of CD8+ T cells with MHC multimers and antibodies against intracellular cytokines • ICS relies upon stimulation of T cells in presence of an inhibitor of protein transport, thus retaining cytokines inside the cell • ICS staining on customer samples available as service from ProImmune © Copyright ProImmune Limited 2011. All Rights Reserved Intracellular Cytokine Staining Protocol (1) Stain cells with Pentamer Stimulate 1 hour with specific peptide / alternative Add Inhibitor of protein transport, e.g. Brefeldin A Culture 37°C for 5-24 hours © Copyright ProImmune Limited 2011. All Rights Reserved Intracellular Cytokine Staining Protocol (2) Stain cells with any additional cell surface markers (optional) Fix with 4% Paraformaldehyde Permeabilize cells Stain with antibodies to Intracellular markers, e.g. antiIFNg Fix with formaldehyde for analysis © Copyright ProImmune Limited 2011. All Rights Reserved ICS / Pentamer Staining Data Activation Controls Gated on Live, CD8+ cells B*08:01/RAKFKQLL (EBV BZLF-1) Stimulation with PMA Pentamer R-PE 1.79% 2.01% 20.99% IFNg - FITC 30,000 live Human PBMCs 10ul Pentamer CD8 clone HIT8a IFNg clone 4S.B3 © Copyright ProImmune Limited 2011. All Rights Reserved B*08:01/RAKFKQLL (EBV BZLF-1) No stimulation 3.39% 0.04% 0.01% ICS / Pentamer Staining Data Stimulation with peptide Gated on Live, CD8+ cells B*08:01/RAK Pentamer (RAKFKQLL peptide) Pentamer R-PE 1.48% 0.52% 0.05% IFNg - FITC 30,000 live Human PBMCs 10ul Pentamer CD8 clone HIT8a IFNg clone 4S.B3 © Copyright ProImmune Limited 2011. All Rights Reserved Negative control A*02:01/GLC Pentamer (RAKFKQLL peptide) 0.06% 0.01% 0.58% Importance of Staining with Pentamer Prior to Activation B*08:01/RAK Pentamer (RAKFKQLL peptide) Staining pre-activation Pentamer R-PE 1.48% 0.52% 0.05% IFNg - FITC 30,000 live Human PBMCs 10ul Pentamer CD8 clone HIT8a IFNg clone 4S.B3 © Copyright ProImmune Limited 2011. All Rights Reserved B*08:01/RAK Pentamer (RAKFKQLL peptide) Staining post-activation 1.10% 0.28% 0.46% MHC Pentamer & Intracellular Cytokine Staining (ICS) Tellam et al. (2007) J. Exp. Med 204: 525-532 Protein translation affects the molecular dynamics of antigen presentation B*08:01 / FLRGRAYGL Pentamer - R-PE EBNA1 EBNA1-DGA 35.1% 50.3% IFNg - FITC • Comparison of immune responses to EBV encoded nuclear antigen with (EBNA1) and without (EBNA-DGA) internal Gly-Ala repeat sequence • EBNA-DGA has more rapid protein translation efficiency • Increased frequency of EBNA-1 specific and functional T cells were observed in the EBNA-DGA version © Copyright ProImmune Limited 2011. All Rights Reserved Conclusions • Most consistent MHC Multimer Technology • Pentamer staining is highly flexible • Pentamers can be used in combination with other cell surface and intracellular markers • Pentamers can be used to isolate antigen-specific cell populations • Results of Pentamer staining can be combined with other techniques, (e.g. ELISPOT) to gain a full picture of an antigenspecific response © Copyright ProImmune Limited 2011. All Rights Reserved CD1d Tetramers - Introduction • Natural killer T (NKT) cells implicated in regulation of immune responses associated with a broad range of diseases • Unique lymphocyte population that co-express NK cell markers and a semi-invariant T cell receptor • When stimulated with CD1d-restricted glycolipid antigen, NKT cells produce Th1-type and/or Th2-type cytokines • CD1d are non-classical MHC molecules, characterized as nonpolymorphic and conserved among species • Possess binding pockets capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells • Best characterized CD1d ligand is α-Galactosyl Ceramide* (α-GalCer) © Copyright ProImmune Limited 2011. All Rights Reserved CD1d Tetramers • CD1d-lipid complexes bind to T cell receptors of NKT cells • Allow identification and enumeration of NKT cells by flow cytometry • Excellent brightness and experimental reproducibility • Several options regarding fluorescence and loading • Only commercial source worldwide for fluorescently labeled human and mouse CD1d tetramers © Copyright ProImmune Limited 2011. All Rights Reserved CD1d Tetramers – Available Options • Loading options − Pre-loaded with alpha-Galactosyl Ceramide* for convenience − Negative Control (mock-loaded with carrier only) − Also supplied empty for loading with the ligand of your choice • Mouse or human CD1d • R-PE or APC labeled * The alpha-GalCer molecule and derivatives thereof are covered by US Patent No.5,936,076, which is owned by Kirin Pharma. The alpha-GalCer molecule is licensed to Funakoshi Co. Ltd. for research use worldwide. © Copyright ProImmune Limited 2011. All Rights Reserved Primary Applications of CD1d Tetramers Flow cytometry Antigen-specific CD1d-restricted NKT cell isolation Intracellular Cytokine Staining © Copyright ProImmune Limited 2011. All Rights Reserved Staining on Human Peripheral Blood Cells Non-specific staining was eliminated by gating on CD19 negative cells before plotting CD1d tetramer vs CD3. Human CD1d APC a-Gal Cer-loaded CD1d tetramer CD3 Human PBMCs 50,000 live events 0.5ul Tetramer CD3 clone UCHT1 © Copyright ProImmune Limited 2011. All Rights Reserved Negative control tetramer Staining on Murine Cells Splenocytes from a naïve B6 mouse depleted of B cells. Data shown is gated on live, CD3 positive cells. Empty tetramer a-Gal Cer loaded Murine CD1d R-PE 0.11% CD4-Alexa 488 Data courtesy of Markus Skold and Sam Behar, Harvard Medical School © Copyright ProImmune Limited 2011. All Rights Reserved 2.5% ProImmune’s Human CD1d tetramer can detect NKT cells from non-human primates R-PE labeled human CD1d tetramers used to stain PBMCs from rhesus macaque monkeys. PBMCs were pre-stimulated with RGI-2001 (liposomal α-GalCer, a ligand for NKT cells) for 3 days. Data shown is gated on live, CD3 positive cells. Stimulated 97.1% Untreated 2.93% Human CD1d R-PE Data courtesy of Omar Duramad, RegImmune Inc. © Copyright ProImmune Limited 2011. All Rights Reserved 99.7% 0.31% CD1d Tetramers - Conclusions • Consistent, reliable technology • Only available from ProImmune • Flexible labeling options • CD1d tetramers can be used in combination with other cell surface and intracellular markers • CD1d tetramers can be used to isolate antigen-specific CD1drestricted NKT cell populations • Results of CD1d staining can be combined with other techniques, (e.g. ELISPOT) to gain a full picture of an NKT cell specific response © Copyright ProImmune Limited 2011. All Rights Reserved