An Integrated Approach to T Cell Immunology

1
An Integrated Approach to T Cell Immunology
BD Biosciences
2
T Cell Immunology Tool KitMethods for Monitoring
Cellular Responses

• DimerX (MHC/peptide binding)
• Intracellular Cytokine/Chemokine Staining
• Antigen Specific Cytokine Flow Cytometry
• ELISPOT
• CBA (Cytometric Bead Array)
• T Cell Signaling Reagents
• IMag Magnetic Separation Reagents

3
T Cell Immunology Tool KitMHC/TCR Signaling
4
MHC Class I and Class II Gene Products

• T cell receptors do not bind to soluble antigen
• Antigen must be presented to the T cells via
  association with MHC by antigen presenting cells
  (Macrophages, Dendritic cells, B cells)
• MHC Class I Gene Products
• Presents antigen to CD8 T cells
• Generates a cytotoxic response
• MHC Class II Gene Products
• Presents antigen to CD4 T cells
• Generates cytokine production, immunoglobulin
  secretion

5
MHCAntigen Processing

• MHC Class I Gene Products
• Soluble antigen in the cytosol from infectious
  agent
• Peptides generated from processing of
  intracellular parasites
• Peptide (8-10 amino acids) bound to appropriate
  MHC molecule in endoplasmic reticulum
• MHCPeptide complex is transported to the cell
  surface by the Golgi apparatus for antigen
  presentation

6
Overview - What is Dimeric Technology?

• Dimeric reagents
• Identifies antigen-peptide specific T cells
• Mimics specific MHC-peptide-TCR interactions
• Expressed as a single recombinant protein
• Easily loaded with peptide of choice at time of
  use (active or passive loading)
• Loaded constructs are stable over time
• Ability to screen peptide libraries

7
Production of DimerX Reagents

• Ig/MHC fusion protein
• Expressed in a mammalian cell line (J558L)
• Produced from J558L cells transfected with a
  plasmid encoding a conformationally intact fusion
  protein.
• Fusion consists of MHC and mouse IgG scaffold
  w/ b2M (beta 2 microglobulin required for MHC
  Class 1 function)

8
Divalent versus tetravalentsoluble MHC analogs
9
DimerX Peptide Loading Protocol

• Passive Loading of peptide
• Mix peptide and DimerX protein together in PBS,
  pH 7.2, incubate for 48 hr. at 4C.
• Peptides should be about 8-10 aa in length and
  high purity for best results
• For high affinity peptides, loading can be done
  in as quickly as one hour using an incubation
  temperature of 37C
• For low affinity peptides, use the longer
  incubation period.
• Unused peptide-loaded DimerX reagent may be
  stored at 4C for up to 1 week.

10
DimerX Staining Protocol

• 1. Resuspend cells in FACS staining buffer or BD
  PharMingen Stain Buffer (FBS)
• 2. Add 0.25 to 4 ?g of peptide-loaded DimerX
  protein to cell suspension.
• 3. Incubate 60 minutes at 4C
• 4. Wash cells 1? with staining buffer.
• 5. Add a fluorescent secondary reagent like
  PE-conjugated anti-mouse IgG1 and Mouse FcBlock
  (for Mouse DimerX reagents)
• 6. Incubate 30 to 60 minutes at 4C.
• 7. Wash cells, resuspend cells in staining
  buffer, analyze by flow cytometry.

11
Quantitation of antigen-specific T lymphocytes
in peripheral blood
HAM
Control
HIV
Tax-A2/Ig
Gag-A2/Ig
CD8
12
Tax-specific CD8 T cells in CSF and PBL in HAM
Tax-A2/Ig
Gag-A2/Ig
CSF
PBL
CD8
13
DimerX Products

• DimerX Available Products
• Mouse H-2KbIg, .25mg (about 200 tests)
• Mouse H-2LdIg, .25mg
• Human HLA-A2.1, 50ug (about 50 tests)
• use between .25ug-4ug/test
• Related Products
• Second Step Reagents
• Anti-Mouse IgG1
• Anti-Mouse CD16/32, Mouse FcBlockTM
• Phenotypic specific reagents (T/NK reagents)
• V? /TCR reagents

14
T Cell Immunology Tool Kit
Traditional Cytokine Flow Cytometry
IL-2 Phycoerythrin
CD4 FITC
15
CFC Staining Protocol - Key Steps
Stimulate and Harvest Cells
Block Fc Receptors
Stain Cell Surface Antigens
Fix and Permeabilize Cells
Stain Intracellular Cytokines
Intracellular Staining Controls
Analyze by Flow Cytometry
16
Selected Activation Methods

• . TCR mediated (CD3/CD28)
• . Mitogenic (PHA/Con A)
• . Polyclonal (PMA/Ionomycin)
• . Lipopolysacharride (LPS)
• . Superantigen (SEB)
• . Antigen specific (Peptides)

17
Cytokine Stimulation Critical Parameters

• Appropriate activation method and kinetics should
  be determined for each experimental system.
• Choice of Protein Transport Inhibitors
• Increased I/C staning signal by enhancing
  accumulation of intracellular cytokines
• GolgiPlug/Brefeldin A
• Disrupts formation of Golgi thus inhibiting
  cytokine secretion
• GolgiStop/Monensin
• Inhibits secretion of cytokines across cellular
  membrane

18
Protein Transport Inhibitors

• GolgiPlug vs. GolgiStop

19
Protein Transport Inhibitors
Golgi Plug/Brefeldin A
GolgiStop/Monensin
No Golgi Inhibitor
20
Protein Transport Inhibitors

• GolgiPlug vs. GolgiStop
• Researchers need to evaluate which is best for
  their system (points of interest)
• Short term activation cultures both equally good
  (mouse/human)
• Comparable in inducing IL-2
• GolgiPlug (Brefeldin A)
• Superior for LPS or PI induced TNF-a analysis
  (mouse/human)
• Superior for IL-6, IL-12, mcp-1, TNF-a analysis
  (mouse)
• GolgiStop (Monensin)
• Mandatory for LPS induced monocyte IL-10 analysis
  (human)
• Superior for IL-4 detection
• Preferred for IL-3, IL-4, IL-5, IL-10, IL-13 in 5
  day restimulation cultures

21
Blocking

• Blocking non-specific binding
• Mouse, and Rat Systems
• FcBlock (CD16/CD32)
• Human Systems
• Heat-aggregated IgG
• Excess of irrelevant purified Ig
• Human serum

22
Stain Surface Antigens

• Buffers formulated to enhance CFC staining
• PharMingenStain Buffer w/FBS
• PharMingenStain Buffer w/BSA
• Useful for sample cell
• mAb dilution
• Cell suspension
• Cell washing
• Cell storage
• Biotin free to reduce background
• Prevents membrane capping

23
Fix and Permeabilize

• Cytofix Buffer
• Cytofix/Cytoperm Kit
• Cytofix/Cytoperm solution
• Perm/Wash Buffer
• Cytofix/Cytoperm Plus with GolgiStop
• Cytofix/Cytoperm solution
• Perm/Wash Buffer
• GolgiStop
• Cytofix/Cytoperm Plus with GolgiPlug
• Cytofix/Cytoperm solution
• Perm/Wash Buffer
• GolgiPlug

24
CFC staining w/ Cytokine and Chemokine Antibodies

• Large portfolio of conjugated
• cytokine and chemokine mAb.
• Compatible with Fix/Perm procedure
• APC and PE give brightest staining (TH2)
• FITC appropriate for more highly expressed
  cytokines (TH1)

25
CFC staining w/ Cytokine and Chemokine Antibodies

• Optimal concentration is very important
• New pre-titrated PE conjugates
• 100 test size
• Optimized for the most effective detection of
  cytokines and chemokines
• General range 0.5ug/test-.015ug/test

26
Blocking Controls for CFC Staining

• Purified Antibody Blocking Controls
• Pre-treat with purified form of mAb to block
  cytokine specific staining.
• Same clone
• 5.0 ug/test optimal

27
Blocking Controls for CFC Staining

• Or...Recombinant Cytokine Protein Blocking
  Controls
• Pre-treat with recombinant cytokine protein to
  block non-specific staining.
• 0.25 ug/test optimal
• Not all recombinant cytokines are effective for
  blocking
• (ie. recombinant mouse and human IL-12 and
  IFN-gamma)

28
Isotype Controls for CFC Staining

• Antibody of same isotype as that being used
• Same idea as for cell surface staining
• Demonstrates inherent non-specific background
  levels
• Negative staining control
• Subtract non-specific staining from from
  positive cytokine staining, these are actual
  positives

29
Cytokine Positive Controls for CFC Staining

• Cytokine/Chemokine Positive Control Cells
• Human, Mouse, and Rat
• Pre-tested for cytokine/chemokine expression
• Express most commonly analyzed cytokines
• IFNg, TNFa, GM-CSF, GRO, IP-10, MCP-1 3, MIG,
  MIP, IL-1a, IL-1?, IIL-2, IL-3, IL-4, IL-6, IL-8,
  IL-10, IL-12, IL-13
• Useful staining controls
• Titer staining concentrations
• Titer blocking concentrations

30
CFC Starter Kits

• Ready-to-use kits for activating and staining
  mouse or human lymphocytes
• Contents
• Buffers
• PE conjugated pre-diluted antibodies
• IL-2, IFN-gamma, and TNF-alpha
• 25 tests each
• Controls
• Positive Control Cells
• Purified Blocking antibody cocktail
• PE Isotype control cocktail
• Detailed Protocols

31
CFC Proliferation
BrdU Flow Kit

• Measures cellular incorporation of BrdU with
  gentle fixation and permeablization at neutral pH
  which allows the concomitant detection of other
  cellular determinants.
• Bromodeoxyuridine (BrdU) is a thymidine analog
• Allows measurement of cell proliferation and
  cell cycle status
• May be used in vitro and in vivo
• BrdU is incorporated into the DNA of cycling
  cells
• Incorporated BrdU is detected with anti-BrdU mAb
• Prolonged exposure identifies cycling cells
• Pulse labeling allows determination of cell-cycle
  kinetics

32
BRDU Flow Kit

• Allows the simultaneous detection and correlation
  of
• S-Phase (FITC)
• Cell Cycle (7-AAD)
• Phenotype (PE, APC, Cy5, Cy5.5)
• Activation Status (PE, APC, Cy5, Cy5.5)
• Nuclear Determinants (PE, APC, Cy5, Cy5.5)
• Cytoplasmic Determinants (PE, APC, Cy5, Cy5.5)
• Cytokines/Chemokines (PE, APC, Cy5, Cy5.5)
• Other.. (PE, APC, Cy5, Cy5.5)

33
BrdU Flow Kit

• Flexible staining protocols
• Procedure can be performed in one day
• 3 hours staining time
• Cells can be fixed and stored
• Wash and store overnight at 40C
• Wash and freeze in cryopreservative solution at -
  800C
• Kit Contents
• FITC anti-BrdU - dilute 150
• Cytofix/Cytoperm Buffer - ready-to-use
• Perm/Wash Buffer - dilute 110
• Cytoperm Plus Buffer (Formulated for BrdU Flow
  Kit)
• BrdU
• DNase
• 7-AAD

34
BrdU Flow Kit
35
CFC Proliferation

• Allows the correlation of
• Phenotype
• Cytokine expression
• Cell Cycle
• Proliferation

36
T Cell Immunology Tool Kit
Antigen Specific Cytokine Flow Cytometry
CD69 PE
anti-TNF? FITC
37
Antigen Specific Cytokine Flow Cytometry

• Simultaneous single cell detection of cell
  surface and intracellular events (e.g. cytokines,
  activation antigens, proliferation, phenotypic
  markers)
• Whole blood (physiological conditions)
• Rapid method (lt6 h)
• Compatible with variety of stimuli including
  antigen

38
Antigen Specific Cytokine Flow CytometryStaining
Procedure
FACSLyse, 10 min
EDTA/PBS
Brefeldin A
1 ml whole blood Ag CD28 CD49d
2h
4h
15 min
Centrifuge, aliquot into staining tubes or freeze
Wash
Wash, fix, analyze
FACS Perm, 10 min
3- or 4-color Flow cytometry
mAb staining, 30 min
39
CD4 Cytokine Responses to Various Antigens
40
Memory Cells Respond to CMV
0.62
0.01
0.03
1.88
17 h
6 h
TNFa PE
1.37
0.06
0.01
0.38
10 h
24 h
CD45RO FITC
41
Differences in CD4 CMV Responses in 3 Donors
42
Gating is Critical to Results
43
Kinetics of CD4 Responsesto CMV
44
The Dump Channel Concept

• Stain with a CD33/CD62P APC cocktail to identify
  monocytes and activated platelets
• Acquire with a logical gate
• CD4 vs SSC R1
• CD33CD62P vs FSC R2
• Analyze FL1 vs Fl2 with a G1 (R1 and not R2)
  logic gate

45
Addition of a Dump Channel
No stimulation CMV stimulation
Without dump With dump
CD69
IFNg
46
HIV Antigen Responses(CD4 Gated)
native HIV-1
recombinant p55
1.24
0.91
CD69 PE
anti-IFN? FITC
47
CD4 response to p55 Gag
A. Long-term non-progressors B. Potential
non-progressors C. Active infection/ progressive
disease D. Short-term HAART E. Long-term
HAART F. HIV- controls
48
CD4 T cell cytokine response to HIV-1 antigen
following 3 immunizations with Remmune
Viral load lt400 CD4 1147
CD69-PE
anti-IFNg FITC
49
HIV-1 immunization enhances Ag-specific CD4 T
cell cytokine responses in seropositive subjects
3.6
1.1
1
IFNg positive CD4 cells
0
Baseline
Week 4
Week 16
Week 28
50
CD4 vs CD8 T cell responses
Ag
APC
CD4 T cell
Whole protein
MHC II
CD4
cytokines
MHC I
peptide
CD8 CTL
T, B, or APC
Optimal peptide
CD8
cytokines
MHC I
51
Use of a Peptide Mix as an Antigen for CFC Assays
15 a.a.
etc.
11 a.a.
CMV pp65 138 peptides
52
Example of CD4 and CD8 responses to CMV pp65
peptide mix
pp65 protein
peptide mix
A2 peptide
CMV lysate
7.41
0.27
0.04
0.27
CD4
CD69 PE
0.19
2.03
1.14
0.87
CD8
anti-IFNg FITC
53
CD8 Cytokine expression is unaffected by
cryopreservation
_______CD4________
________CD8________
8
7
6
5
Fresh
cytokine positive cells
4
Frozen
3
2
1
0
IFNg
TNFa
IFNg
IFNg
IFNg
TNFa
TNFa
TNFa
CMV lysate
CMV lysate
Peptide mix
Peptide mix
54
Flow Cytometric Detection of Cytokine Production
and Cell Proliferation

• 1. Activate PBMC for 48 to 72 hr at 37?C
• 2. Add 60 ?M BrdU for the last 6 hours
• 3. Fix and permeabilize using FACSLyse and FACS
  Permeabilizing solutions (10 min RT each)
• 4. Stain for surface markers (e.g. CD4), cytokine
  (e.g. IFN?) and BrdU DNase
• 5. Analyze by 3 or 4 color flow cytometry

55
Proliferation Cytokine Expression in
CMV-activated CD4 PBMC (48 h)
56
Critical Parameters for Antigen Specific Cytokine
Staining

• Optimized activation protocol
• Costimulatory antibodies (CD28, CD49d)
• Addition of secretion inhibitor (eg. Brefeldin A)
• Standardized fixation and permeabilization
  reagents
• Anti-cytokine antibodies with high affinity for
  intracellular epitopes
• Pure Ab-fluorochrome conjugates (i.e. no free
  dye) to minimize background

57
Technical Essentials

• Use only heparanized blood
• Age of blood (lt24h)
• Use polypropylene tubes or BSA-coated tubes
• EDTA treatment vortex
• Decant (or aspirate very carefully)
• Use only high quality mAb for intracellular
  staining
• Flow analysis - Gate on both FSC vs. SSC and FL3
  (CD4) vs. SSC

58
Cytokine Flow Cytometry Advantages

• Multiple events per cell (cell surface
  intracellular nuclear)
• Multiple cell types identified
• Simple and rapid procedure amenable to automation
• Reproducible, quantitative assay for
  antigen-specific T cell frequency

59
T Cell Immunology Tool KitSingle Cell Assays
ELISPOT
60
ELISPOT vs Ag-specific Cytokine Flow Cytometry

• ELISPOT
• Measures cytokine production at a single cell
  level
• Single cell sensitivity
• Determinant mapping
• Peptide screening
• Minimal cell needed
• Non pharmacologic
• Identifies rare cell events
• Front end HTS

• Ag-specific CFC
• Measures cytokine production at a single cell
  level
• Multi-parameter
• Activation status
• Effector function
• Phenotypic identification
• Proliferation
• Cytokines/Chemokines
• Identifies rare cell events

61
ELISPOT Assay Applications

• Antigen specific immune response
• Vaccination studies, monitoring
• HIV, HBV, HCV monitoring
• Th1/Th2 analysis
• Epitope mapping
• Assessment of cellular immunity, activation
  status
• Stem cell function characterization
• Transplant rejection prediction
• Autoimmune disease characterization
• QC of anti-tumor cell therapies, etc
• Assessment of secretion products of very low
  frequency tissue, tumor, and immune cells

62
Introduction to ELISPOT

• Very similar to ELISA, except
• Add live cells to microwells, not supernatants,
  serum, plasma, lysates.
• Purified or unpurified cells activated in
  microwells
• Results are spots, yielding frequency analysis of
  producer cell population (frequency analysis
  similar to flow cytometry, without surface
  antigen phenotyping)
• Single or dual parameter (NTB and AEC)

63
Introduction to ELISPOT

• Assay Principles
• Each reactive cell (e.g., specific
  analyte-producer) leaves a mark as a colored
  spot, usually 10-20 µM.
• Number and size of spots reflects reactivity of
  immune system, for example.
• Exquisitely sensitive means of elucidating
  effector cell frequency by secreted products
  (e.g., Th 0/1/2/3)

64
ELISPOT PROCEDURE
65
ELISPOT - Procedure

• Aseptically -
• Coat microwells of ImmunoSpot plate with
  anti-cytokine capture antibody (e.g., PBS or
  other buffer 1-6 µg/ml 100 µl O/N, 4?C)
• Block microwells with complete medium including
  5 FCS (1 hr, RT)
• Add fractionated or unfractionated cells (102 -
  106 cells/100µl) are added to microwells with
  specific antigen, peptide or mitogen, etc. (2-24
  hrs, 37?C)

66
ELISPOT - Assay procedures

• On the bench
• Rinse plate with dH2O
• Wash plate with PBS-Tween
• Add biotinylated detecting antibody, diluted in
  PBS-Tween (100 µl, O/N, 4?C). Wash plate with
  PBS-Tween
• Add Sav-Enzyme (e.g., HRP, AKP 2 hr 4?C). Wash
  - PBS-Tween
• Add visualization solution / insoluble substrate
  (e.g., AEC, NBT/BCIP). Wash thoroughly in dH2O.
  Air dry store protected from light
• Scan and count on ImmunoSpot Analyzer

67
ImmunoSpot Plates
68
ImmunoSpot Plates

• Opaque white - optimized for automated image
  analysis
• PVDF high protein binding membrane
• Maximal signalnoise
• Unique high sensitivity Choice of working
  volumes
• 200 µl or 50 µl
• Batch tested on CTL Analyzers

69
Standard ELISPOT plates vs. ImmunoSpot plates
70
ImmunoSpot Series I Analyzer
71
Unique Features ImmunoSpot Series 1 Analyzer

• Fast, fully automated acquisition and storage of
  high resolution images on CD or workstation
• Auto-centering of wells during plate scan
• Background balance for dealing with complex
  results
• User-definable counting parameters
• Fully automated image analysis
• Custom imaging for presentations
• Versatility, expandability of system

72
Automated Image Acquisition and Storage

• ELISPOT microtiter plate is read in fully
  automated mode
• High-resolution images saved as individual wells
  and composite images of whole plates to CD or
  ImmunoSpot Satellite.
• Each CD can store 864 detailed well images (667
  KB each) for high-resolution single cell analysis
• Stand-alone system is equipped with camera, lens,
  illumination, high precision X-Y stage.
  Microscope not required.

73
Auto-centering of wells during plate scan

• Unique, automatic, well-centering capability
• Does not require manual corrections
• Overcomes common irregularities in plate
  manufacturing
• Enables automation/high-throughput

74
Background balance

• Eliminates artifacts from
• Uneven illumination of wells
• Irregular background staining
• Membrane leakages
• Enables counting of spots on light and dark areas
  with same counting parameters

75
User-definable counting parameters

• Sensitivity - select spots by color density
• Morphology - enables counting of wide range of
  spots
• Small/dense to big/diffuse
• Separation control - enables identification of
  closely situated spots
• Spot size gating - enables gating by size,
  histogram data
• Overdeveloped area handling
• Enables counting of overdeveloped and/or
  overcrowded areas
• Enables counting of wells with high background

76
BD PharMingen Cytokine ELISPOT Reagent Sets

• Include
• 10 ImmunoSpot M-200 plates
• Unlabelled capture antibody (0.5 mg for 10
  plates)
• Biotinylated detection antibody (0.25 mg for 10
  plates)
• Sav- HRP
• Developed in collaboration with CTL LLC

77
PharMingen ELISPOT Reagent Sets

• Available now
• Mouse IFN-?, IL-2, IL-4, IL-5
• Human IL-4 IL-5, IL-10
• Available Summer 2001
• Mouse TNF-?,
• Human TNF-?, IL-2
• Available Fall 2001
• Human IFN-?

78
ELISPOT Assay - Unique Strengths

• Highly sensitive frequencies below 1300,000
  cells
• Minimal material needed for analysis. E.g., 20
  mls blood for analysis of immune responsiveness
  to 700 peptides.
• Non-pharmacologic. Golgiplug (Brefeldin A) not
  needed.
• Assesses functionality.
• High throughput, 96 well plate, high content
  analysis. 30 plate auto loader or robotics
  options.
• Compatible with other assays. Run ELISPOT for
  cytokines, then transfer cells for cloning or
  other assays (e.g., proliferation assay).

79
T Cell Immunology Tool Kit
Cytometric Bead Array (CBA)
80
Multiplex Bead Assays by FlowBeads are
Distinguished by Size or by Color
Multiple Sizes
Shades of a color
Combined shades of colors
81
BD CBA System

• Reagent Kits
• Mouse Immunoglobulin Isotyping Kit
• Human Th1/Th2 Lymphokine Kit
• Mouse Th1/Th2 Lymphokine Kit
• BD CBA Software
• Excel Plug-In (Macintosh)
• Accepts FCS 2.0 Files
• Flow Cytometer Setup System
• Cytometer Setup Beads

82
CBA

• Reagent Kits
• Mouse Immunoglobulin Isotyping Kit
• 100 samples
• IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, IgA
• Kappa/Lambda
• Ascites and Supernatants
• Human Th1/Th2 Kit
• 50 samples
• IFN-?, TNF-?, IL-2, IL-4, IL-5 ,( IL-6), IL-10
• Supernatants and Serum
• Mouse Th1/Th2 Kit
• 50 samples
• IFN-?, TNF-?, IL-2, IL-4, IL-5
• Supernatants and Serum

83
BD CBA Software
Toolbar
Home Screen
84
Load Standards Data Files Enter Concentrations
85
Automated Calculation of Standard Curves
86
Load Sample Data FilesEnter Dilution Factors
87
Human Th1/Th2 CBACytokine Panel Assay

• Th1/Th2 panel IL2, IL4, IL5, (IL-6), IL10, TNF?,
  IFN?
• 6 (7) beads, dyed to different FL3 intensities
• FL-2 (PE )signal generator
• Software assisted analysis, with curve fitting
  algorithm
• Result reporting included in software

88
One Bead Size6 - FL3 Intensity Levels













89
CBA Assay Format

• 6 cytokines from a 50 ?L sample
• Three pipetting steps
• 180 minutes total incubation
• Homogeneous no-wash or single wash assay
• Acquire 300 events/bead (6 bead assay-collect
  1800 events
• Target sample is activated cell supernatant or
  serum

90
Assay Range Analytical Sensitivity
Analyte Assay Range Analytical
sensitivity IFN ? 0 - 5000 pg/ml 7 pg/ml
TNF? 0 - 5000 9 IL 10 0 - 5000 11 IL
5 0 - 5000 10 IL 4 0 - 5000 13 IL 2 0
- 5000 6
Sensitivity Mean (N6) 2 S.D. of zero
calibrator
91
Comparative StudyMultiplex Bead Assay vs. ELISA
Analyte Correlation Coefficient N IFN ?
0.98 9 TNF? 0.97 20 IL 10
0.99 20 IL 5 1.00 10 IL 4 0.94
20 IL 2 0.98 17 Samples
Supernatants from cultured human PBMCs
92
Human Th1/Th2 CBA Raw Data
Human PBMC Treated for 24 Hours with PMA/Ionomycin
93
Human Th1/Th2 CBA Software Analyzed Data
Human PBMC Treated for 24 Hours with
PMA/Ionomycin
94
Application of Th1/Th2 panel
Effect of IL2 treatment on cytokine profile
IL2 Spiked Pre-Treatment Serum
Before Treatment
After Treatment
IL-2 IL-4 IL-5 IL-10 TNF? IFN?
95
CBAAssay Benefits

• Measure multiple analytes, Small sample volume
  needed
• High throughput
• No artifacts from enzyme generates signal (ELISA)
• Little hands on time
• FACScan and FACSCalibur with Loader (FACSVantage
  SE, BD-LSR, and Coulter XL can also be used)

96
T Cell Signaling
97
IMag Magnetic Particles

• Streptavidin magnetic particles
• 0.1 to 0.45 microns
• Covalently coupled to streptavidin
• Optimized for use with High Gradient Magnetic
  Separation Columns
• Paired with biotinylated mAb

98
IMag Magnetic Particles

• Method
• Adjust single cell suspension to 20 million
  cells/ml
• Add Fc Block 0.25 ug per million cells
• Add biotinylated mAbs
• Incubate at 4ºC for 15 minutes
• Wash with 15 x volume of IMag buffer
• Add 10ul Imag particles per 10 million cells
• Incubate at 4ºC for 30 minutes
• QS to 500ul load column

99
IMag Magnetic Particles

• Sets
• Mouse CD4
• Mouse CD8a
• Mouse CD11b
• Mouse CD45R/B220
• Mouse CD90.2
• Mouse Ly-6G
• Available Spring 2001

100
T Cell Immunology Tool KitMethods for Monitoring
Cellular Responses

• DimerX (MHC/peptide binding)
• Intracellular Cytokine/Chemokine Staining
• Antigen Specific Cytokine Flow Cytometry
• ELISPOT
• CBA (Cytometric Bead Array)
• T Cell Signaling Reagents
• IMag Magnetic Separation Reagents