Transcript 1. dia - Department of Immunology

Measuring the functional activity of
T and B lymphocytes
Polyclonal activation of T and B cells
lectin-induced activation
α-IgM, α-CD3 or α-TCR antibody
allogeneic T cell activation
(examination of the immediate-early activation events)
T and B cell response
activation markers
proliferative response:
3H-thymidine
incorportion
CFSE fluorescence decrease
cell cycle events
Antibody or cytokine production (ELISA, bioassay, CBA)
Determinating the number of activated T and B cells after the
administration of the antigen
ELISPOT,
Intracellular cytokine staining
MHC tetramers
(review)
Phases of the humoral immune response
(review)
Phases of T cell response
Immunodeficiencies mainly characterized by different
functional immunoassays
Lymphocyte activation by specific antigen is hardly detected,
because of the low number of the antigen specific cells
Lymphocyte function can be investigated by polyclonal T/B-lymphocyte
activator materials
Polyclonal activation of lymphocytes by
LPS, lectins, PMA/ionomycin
BAcell
TBcell
TCcell
TLR4
(PMA activates protein kinase C)
BCR or TCR-specifc antibodies could activate the lymphocytes also
Polyclonal B cell activators
Activator
T cell dependency
Ig secretion
Human B cells
PWM (pokeweed mitogen)
no
yes
SpA (superantigen, staphylococcus protein A)
no
yes
EBV (transforming effect)
yes
yes
Anti-Ig
yes
In the presence of cytokines
Polyclonal T cell activators
Phytohaemagglutinin (PHA)
lectin
Canavalia ensiformis
Concanavalin A (ConA)
lectin
Phaseolus vulgaris
anti-CD3
Monoclonal antibody
Pokeweed (PWM)
(Phytolacca americana) – formerly
used for coloring red wine
(toxic: triterpene saponin)
Chenopodiales
Phytolaccaceae
Phytohaemagglutinin (PHA)  Canavalia ensiformis – Jackbean, Sword bean
Concanavalin A (ConA)  Phaseolus vulgaris – bean
EXAMINATION OF
T AND B CELL FUNCTIONS
Receptor crosslinking
(immediate)
phosphorylation steps
- Western blot
- Bead array
(seconds-minutes)
Antigen receptors (TCR, BCR), and different
other receptors (eg. cytokine receptors)I.c Ca2+ increase
- FACS, microscopy
Gene activation
- RT-PCR
Cytokine synthesis - i.c cytometry
Cytokine secretion
Cell-cycle/apoptosis
Lymphocyte activation
Cell division
- ELISA, ELISPOT
- Bioassay
- DNA content
- IN antigens
- 3H-thymidine, CFSE, MTT
The examination often requires specific
Ag-Ab reactions
Western blotting
Steps:
1.
2.
3.
4.
5.
sample preparation (cells, tissues)
gel electrophoresis
blotting
labeling
development
Anode(+)
use:
Cathode(-)
Identification of defined components from
protein mixtures by antigen specific
antibodies
Western Blot
• SDS-PAGE gel  resolved into single protein bands (overlap possible)
• Presence of a protein is determined by hybridizing the proteins, transferred or
applied to a membrane, with the relevant antibody
Protein
Standard sample
Antibody recognizes epitope in
specific protein
SDS-PAGE
Membrane
Western blot
Western Blot
• Used to detect specific proteins
in a sample
• Proteins separated by Sodium
Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis (SDSPAGE), transferred to a
membrane
• Primary (1st) antibody
(monoclonal or polyclonal) used
to detect protein
• Enzyme linked 2nd antibody (e.g.
horseradish peroxidase-linked)
used to detect 1st antibody
Investigation of the presence or absence of
Bruton’s tyrosine kinase (BTK) by Western blot
X-linked agammaglobulinemia. XLA patients do not generate mature B cells,
which manifests as an almost complete lack of antibodies in their bloodstream.
Investigation of the presence or absence of
Bruton’s tyrosine kinase (BTK) by flow cytometry
Futatani T et al. Blood 1998;91:595-602
Detection of intracellular Ca2+ concentration
An increase in cytoplasmic
Ca2+ levels can be detected by
fluorescent indicator dyes.
/Fluo-3 or Indo-1/
Fluo-3 AM – excitable by blue light
Indo-1 AM – excitable by UV light
These indicator dyes bound to apolar groups (e.g. acetoxy-methylester: AM) cross the cell
membrane, in the cell, esterases cleve them so the fluorochromes become polar and are
trapped in the cell
e.g. Fluo-3
or
Indo-1
Fluorescence proportional
with Intracellular Ca2+ level
Measurement of Ca2+ signal by flow cytometry
activation
of cells
time
basic signal
You can detect by fluorimeter also
Measurement of Ca2+ signal by flow cytometry
T cell hybridoma specific for influenza virus hemagglutinin protein-derived
peptide - Ca2+ signal by antigen presentation
activated T cells
T cell activation
(APC - T cell)
non-activated T cells
Immunohistochemistry
Labeled antibodies added to fixed tissue sections detect
the distribution of the chosen antigen within the tissue or
within the cells of a particular tissue
• Immunofluorescence
•Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate (green)
PE – phycoerythrin (orange)
• Immunoenzyme method
• enzyme-coupled antibody
P – peroxidase
PA – alkaline phosphatase
(Substrates converted into an insoluble compound)
Immunohistochemistry
Fixation
Tissue
sample
Freezing
Sectioning
Section before
staining
Immunohistochemistry
ABC Method
Enzim
X
Avidin
Biotin
Secondary antibody
Primary antibody
Slide
Cells
Tissue
sample
Classical histochemistry
Acute bronchopneumonia (hematoxilin- eosin staining)
Immunohistochemistry
(CD68+ macrophages and lymphocytes, granuloma)
Antinuclear autoantiboies (ANA) from the serum of a SLE patient can be visualized in cell culture
(Hep-2) by indirect fluorescent labeling (immunofluorescence)
Immunohistochemistry using fluorescent detection
Detection of actin microfilaments
A fixed and permeabilized skin fibroblast.
Mitochondria were labeled with mouse IgG (anti–OxPhos Complex V) and visualized
using goat anti–mouse IgG conjugated with orange-fluorescent Alexa Fluor 555.
F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (a mushroom toxin).
Nucleus was stained with TO-PRO-3 iodide.
Peroxisome labeling in fixed and permeabilized pulmonary artery endothelial cell.
Peroxisomes were labeled using an antibody directed at peroxisomal membrane
protein 70 and detected with Alexa Fluor 488–labeled goat anti–mouse IgG.
Mitochondria were stained with MitoTracker Red prior to fixation;
Nuclei were stained with blue-fluorescent DAPI.
Flow cytometry
An immunofluorescent method that mutually
complements the fluorescent microscopy
• Investigation of different cells or particles travelling high
velocity in flow
• Detects fluorescence intensity and scattered light of the
labeled cells
• Can investigate enormous number of cells in short period
of time
Why flow cytometry
Most cells in the immune system can be found in free or loosely adherent form.
They can be easily suspensed and labeled by fluorescent antigen specific antibodies,
and then they can be examined cell by cell.
The cells’ light scatter and immunofluorescent properties can be analyzed
statistically (eg. percentages of different cell populations)
Rare cell populations can be identified and examined (eg. antigen specific
lymphocytes)
The method provide qualitative and quantitative data – it can detect the presence of
different antigens in the cell, and the expression levels of these antigens. Changes in
the expression of certain molecules can be followed after different treatment of the
specimen. (eg. cell activation, disease progression)
Benchtop flow cytometer
Sorter - flow cytometer
(FACS station)
Example Chanel Layout for Laser-based Flow Cytometry
The emited fluorescent light can
be separeted to components by
special mirrors and filters
photodetectors
PMT 4
cell
suspension
in tube
flow cell
PMT 3
PMT 2
PMT 1
forward light scatter
detector
Laser
(PMT=photo-multiplayer tube)
Light scatter and fluorescence
Laser
Forward angle
light scatter
sensor
(FSC, FALS)
Can be loosely
considered as a
representation of the
particle size
Side light scatter(SSC) and
fluorescence detectors
represents the granularity of
the cells
Multocolor staining can be used to
identify cell sub-populations
* autofluorescence – presence of piridins and flavins.
Immunophenotyping
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(eg. monitoring AIDS progression)
Labeling:
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Th
NK
Tc
Lymphocytes in the periferial blood
Fluorescent microscopy
B
high velocity flow stream
detecting CD4-FITC
labeled (TH) cell
(in cuvette or stream in air)
detector
signal
processing unit
CD8
PE
screen
increasing light
intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
microscopy:
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal
processing unit
increasing light
intensity
CD4
FITC
detecting the unlabeled cell
(eg.B cell) by autofluorescence
CD8
PE
detector
Signal
processing unit
increasing light
intensity
microscopy:
dim (autofluorescent)
cell
CD4
FITC
CD8
PE
18%
44%
0%
quadrant
statistics
CD4 38%
FITC
Graphical representations 1.
dot-plot
contourplot
densityplot
Graphical representations 2.
Histogramm
Numeral intensity
values:
~7
~ 1300
homogenous cell population
is normally distributed
(Gaussian)
Different cell types - characteristic light scattering
granulocytes
side light scattering (SSC)
(e.g. granulated)
monocytes
lymphocytes
forward light
scattering (FSC)
(„size”)
Examination of peripheral blood by haematology automats
Measured parameters:
peroxydase staining (the presence of myeloperoxydase, x – axis)
light scatter (high on large granular cells, y – axis)
1 Noise
2 Nucleated Red Blood Cells
3 Platelet Clumps
4 Lymphocytes and Basophils
5 Large Unstained Cells
6 Monocytes
7 Neutrophils
8 Eosinophils
Only the major cell types can be identified
Characterisation of immune cells using cell
surface markers
Cell types, differentiation stages can be identified using a
combination of cell surface markers.
Used in diagnostics:
- ratio of different cell types
- altered expression of cell surface markers
Examples:
- Inflammatory processes – increased neutrophil numbers
- HIV progression – decrease of CD4+ T cell count
CD4+ : CD8+ = 1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
- increase of CD5+ B cells – typical for some B cell Leukemias
Diagnosis of immunodeficiency by flow cytometry
WAS: Wiscott-Aldrich Syndrome
A typical symptom: Lacking or decreased
CD43 expression
XLA: X-linked Agammaglobulinemia
Inhibited B cell development: Lack of CD19+ B cells
Intracellular cytokine detection by
immunofluorescence
cytokine specific antibody with fluorescent labelling
- the cell membrane should be permeabilized (detergent)
- the cells should be fixed previously avoiding the
decomposition of the cells (e.g. aldehyde fixation)
- optionally the cells could be
labelled by some cell type specific
antibody in the beginning (e.g. CD4)
cytokines
One can determine which cell type produced the
cytokines!
ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
well
enzyme linked
immune sorbent
enzyme
Antibody conjugated with
enzyme
Antigen/antibody
adsorbed to solid surface
Enzyme activity in ELISA is directly
proportional to the amount of antigen present
Enzyme activity is measured by the color
reaction due to conversion of substrate
Similar principle applies to many other antibody-based
detection methods
Basic setups in ELISA,
immunohistochemistry, flow cytometry
Indirect method
Direct method
Label
Label
Secondary
antibodies
Primary
antibodies
Antigen
Steps of combined sandwich ELISA
For antigens present at low concentration in complex biological samples
Coating with Agspecific „capture”
antibody
Blocking free plastic
surface with inert
protein
Addition of antigencontaining solution
Addition of biotinylated
antibody specific to a
different epitope on
target protein
Addition of avidinconjugated enzyme
Addition of substrate
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Removal of unbound material
May be you have already met such kind diagnostic tools, or you are
going to meet them during your career
Eg. detection of human chorionic gonadotropin in serum or urine
(pregnancy test)
The principles of these tools are similar as the ELISA
assay you have met before.
hCG Rapid One-Step Immunochromatographic Assay strip
front view
side view
absorbtion pad (cellulose)
control antibody lane
(detection antibody capture)
hCG capture antibody lane
nitrocellulose membrane
(signal detection pad)
glass fiber membrane with visually labeled detection antibodies
sample application pad
urine
detection antibody
capture antibodies
control
antibody line
hCG capture
antibody line
detection antibodies
hCG
hCG +
control line (C)
test line (T)
hCG negative
Competitive system
hCG positive
control line
detection antibody
capture antibody
(
hCG line
bound hCG)
control line
test line
hCG negative
ELISA plates - results
ELISPOT
Enzyme Linked Immuno-Spot
The principles are similar to ELISA
Capable to determine the number of cells that produce
Ig, cytokines, chemokines, granzymes and other soluble
effector molecules
The sensitivity allows the determination of 1 activated
cell among 300,000 others, so it can reveal activated
effector cells not only after polyclonal but after antigen
specific activation
The first steps should be done in aseptic conditions
ELISPOT
The process
- coating with antigen specific capture antibodies
- blocking
- administration of the cells (activation, incubation)
- washing
- administration of biotin conjugated
antigen specific secondary antibody
- avidin-enzyme conjugate
Upper view of a well on
an ELISPOT plate with
- administration of the unsoluble
the generated spots
chromogenic substrate (AEC 3-amino-9-ethylcarbazol)
A spot showing the
place of the cytokine
producer cell
It can be evaluated by microscopy (slow, manual process)
or you can use “ELISPOT plate reader” (fast + standardizable spot
number and size determination)
Bioassay
Very sensitive cytokine determination can be achieved by cytokine
sensitive or cytokine dependent cell lines. The presence or absence
of cytokines determines the fate of the indicator cells that could be
cell proliferation or cell death.
CTLL2 cell line
IL-2 is present
no IL-2
TNF is present
no TNF
Wehi 164 cell line
Living cells can be visualized by colorimetric assay (eg. MTT), or
their proliferation can be measured by other methods.
MTT assay living cells convert the stain to purple-blue
cytokine concentration
IL-2
CTLL2
TNF
Wehi 164
Bioassays could be equivocal, because of the cytokine cross reactivity of indicator cells:
eg. the IL-2 dependent CTLL2 cells can proliferate in the presence of high IL4
concentration also.
Cytokine array
Multiple cytokines can be detected rapidly
in the same procedure
(The process is similar to the procedures of Western-blot after the blotting step)
multiple antigen specific antibodies bound to membrane
„Luminescent antibody” mixture
(+)
IL-2
IL-4
unknown cytokine
containing solution
IL-5
…
IL-12
IFN
…
(
-)
Disadvantage – defined volume sample needed to cover the surface of the membrane
cytokine production of
moDC activated by CD40L and
CD40L+SLAM combination
Réthi és mtsi. 2006
Investigation of gene activation
Activation of T cells can be monitored by the detection
of the transcribed mRNA of the activated genes.
e.g. activation of cytokine genes
method: RT-PCR, QRT-PCR
cells  RNA isolation
RNA  (reverse transcriptase)  cDNA
cDNA  (PCR)  determination of the length and quantity
RT-PCR: agarose gel (densitometry)
QRT-PCR: fluorescent method
(TaqMan probe (FRET) or dsNA intercalating fluorochrome  SYBR green)
PCR
Cell separation
Physical isolation of the cells of interest from a heterogeneous population
Differences in the physical , biological or immunological properties of the cells
are utilized to separate the cells
(differences in cell surface receptor expression is often available – there is a possibility to further
investigate the separated living cells )
physical – density, size
cell biological – adherence, phagocytosis, sensitivity to the
medium
immunological – antigen differences (surface!)
Characteristics of the separation:
• purity
• recovery, yield, lost
• viability of the cells
Separation
Base strategies:
positive separation – labeling and separation of the cells of interest
eg. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody.
The cells become affected both by the separation environment and the
antibodies bound to the receptors. The purity of the separation is generally
high.
negative separation – get rid of the labeled unwanted cells (depletion)
The cells become affected only by the separation environment This is the
preferred strategy in the functional examinations.
The different density parts of the anticoagulated blood has been separating to three parts
in undisturbed tube:
bottom: sedimented red blood cells
top: cell free plasma
the intermediate layer is called „buffy coat” contains the leukocytes, platelets
The process can be accelerated by centrifugation
(from Google pictures)
(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
Ficoll-Paque: density based cell separation
peripheral blood
pipettig the „ring”
containing the
mononuclear cells to a
new tube
centrifugation
to get rid of ficoll
plasma
mononuclear
cells
(PBMC)
ficoll
neutrophil
granulocytes
pipetting cells on ficoll
Red blood cells
separated cells
Magnetic immunoseparation (MACS)
antigen specific antibody
paramagnetic
bead
MACS
Magnetic cell separation (MACS)
separation of labeled cells
(positive separation)
MAGNET
MAGNET
column
depleting or selecting
unlabeled cells
(negative separation)
Magnetic column