Measuring the functional activity of T and B lymphocytes

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Measuring the functional activity of T and B
lymphocytes
Polyclonal activation of T and B
cells lectin-induced activation a-IgM, a-CD3 or
a-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
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(review)
Phases of the humoral immune response
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Phases of T cell response
(review)
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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
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Polyclonal activation of lymphocytes by LPS,
lectins, PMA/ionomycin
T cell
C
T cell
B
A
B cell
TLR4
(PMA activates protein kinase C)
BCR or TCR-specifc antibodies could activate the
lymphocytes also
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Polyclonal B cell activators
Ig secretion
T cell dependency
Activator
Human B cells
yes
no
PWM (pokeweed mitogen)
yes
no
SpA (superantigen, staphylococcus protein A)
yes
yes
EBV (transforming effect)
In the presence of cytokines
yes
Anti-Ig
Polyclonal T cell activators Polyclonal T cell activators Polyclonal T cell activators
Phytohaemagglutinin (PHA) lectin Canavalia ensiformis
Concanavalin A (ConA) lectin Phaseolus vulgaris
anti-CD3 Monoclonal antibody
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Pokeweed (PWM) (Phytolacca americana) formerly
used for coloring red wine (toxic triterpene
saponin) Chenopodiales Phytolaccaceae
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Phytohaemagglutinin (PHA) ? Canavalia ensiformis
Jackbean, Sword bean
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Concanavalin A (ConA) ? Phaseolus vulgaris bean
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EXAMINATION OF T AND B CELL FUNCTIONS
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Receptor crosslinking (immediate)
phosphorylation steps (seconds-minutes)
- Western blot - Bead array
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

• ELISA, ELISPOT
• Bioassay

Cytokine secretion
- DNA content - IN antigens
Cell-cycle/apoptosis
Lymphocyte activation
Cell division
- 3H-thymidine, CFSE, MTT
The examination often requires specific Ag-Ab
reactions
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Western blotting

• Steps
• sample preparation (cells, tissues)
• gel electrophoresis
• blotting
• labeling
• development

Anode()
use Identification of defined components from
protein mixtures by antigen specific antibodies
Cathode(-)
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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 sample
Standard
Antibody recognizes epitope in specific protein
Western blot
Membrane
SDS-PAGE
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Western Blot

• Used to detect specific proteins in a sample
• Proteins separated by Sodium Dodecyl
  Sulfate-Polyacrylamide Gel Electrophoresis
  (SDS-PAGE), 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

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Investigation of the presence or absence
of Brutons 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.
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Investigation of the presence or absence
of Brutons tyrosine kinase (BTK) by flow
cytometry
Futatani T et al. Blood 199891595-602
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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
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Measurement of Ca2 signal by flow cytometry
You can detect by fluorimeter also
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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
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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)

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Immunohistochemistry
Fixation
Sectioning
Tissue sample
Section before staining
Freezing
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Immunohistochemistry ABC Method
Enzim
Avidin
X
Biotin
Secondary antibody
Primary antibody
Slide
Cells
Tissue sample
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Classical histochemistry Acute bronchopneumonia
(hematoxilin- eosin staining)
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Immunohistochemistry (CD68 macrophages and
lymphocytes, granuloma)
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Antinuclear autoantiboies (ANA) from the serum of
a SLE patient can be visualized in cell culture
(Hep-2) by indirect fluorescent labeling
(immunofluorescence)
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Immunohistochemistry using fluorescent
detection Detection of actin microfilaments
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A fixed and permeabilized skin fibroblast.
Mitochondria were labeled with mouse IgG
(antiOxPhos Complex V) and visualized using
goat antimouse 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.
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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 488labeled goat antimouse IgG.
Mitochondria were stained with MitoTracker Red
prior to fixation Nuclei were stained with
blue-fluorescent DAPI.
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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

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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)

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Benchtop flow cytometer
Sorter - flow cytometer (FACS station)
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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
PMT 3
flow cell
PMT 2
PMT 1
Laser
forward light scatter detector
(PMTphoto-multiplayer tube)
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Light scatter and fluorescence
Forward angle light scatter sensor (FSC, FALS)
Laser
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.
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Immunophenotyping
Example
Measurement of CD4 (helper) and CD8 (cytotoxic)
T cell ratio (eg. monitoring AIDS progression)
Labeling
FITC labeled anti-CD4 antibody(a-CD4-FITC) PE
labeled anti-CD8 antibody (a-CD8-PE)
B
NK
Th
Tc
Lymphocytes in the periferial blood
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high velocity flow stream (in cuvette or stream
in air)
detecting CD4-FITC labeled (TH) cell
signal processing unit
screen
detector
CD8 PE
focused laser beam
CD4 FITC
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detecting the PE labeled cell (CD8-PE)
signal processing unit
detector
CD8 PE
CD4 FITC
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detecting the unlabeled cell (eg.B cell) by
autofluorescence
Signal processing unit
detector
CD8 PE
CD4 FITC
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0
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CD8 PE
quadrant statistics
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CD4 FITC
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Graphical representations 1.
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Graphical representations 2.
Histogramm
homogenous cell population is normally
distributed (Gaussian)
Numeral intensity values
7
1300
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Different cell types - characteristic light
scattering
granulocytes
side light scattering (SSC) (e.g. granulated)
monocytes
lymphocytes
forward light scattering (FSC) (size)
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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 Noise2 Nucleated Red Blood Cells3 Platelet
Clumps4 Lymphocytes and Basophils5 Large
Unstained Cells6 Monocytes7 Neutrophils8
Eosinophils
Only the major cell types can be identified
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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 lt200/?l
• - increase of CD5 B cells typical for some B
  cell Leukemias

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Diagnosis of immunodeficiency by flow cytometry
WAS Wiscott-Aldrich Syndrome
XLA X-linked Agammaglobulinemia
A typical symptom Lacking or decreased
CD43 expression
Inhibited B cell development Lack of CD19 B
cells
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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
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One can determine which cell type produced the
cytokines!
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ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
well
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enzyme linked
immune sorbent
Antigen/antibody adsorbed to solid surface
Antibody conjugated with enzyme
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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
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Basic setups in ELISA, immunohistochemistry, flow
cytometry
Direct method
Indirect method
Primary antibodies
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Steps of combined sandwich ELISA
For antigens present at low concentration in
complex biological samples
Removal of excess enzyme
Coating with Ag-specific capture antibody
Removal of unbound material
Removal of unbound protein
Blocking free plastic surface with inert protein
Removal of unbound material
Addition of antigen- containing solution
Addition of biotinylated antibody specific to a
different epitope on target protein
Addition of avidin-conjugated enzyme
Addition of substrate
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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.
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hCG Rapid One-Step Immunochromatographic Assay
strip
side view
front view
absorbtion pad (cellulose)
control antibody lane (detection antibody capture)
nitrocellulose membrane (signal detection pad)
hCG capture antibody lane
glass fiber membrane with visually labeled
detection antibodies
sample application pad
urine
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control antibody line
hCG capture antibody line
detection antibodies
hCG
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Competitive system
control line detection antibody capture antibody
hCG line ( bound hCG)
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ELISA plates - results
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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

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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 the
generated spots
- administration of the unsoluble chromogenic
substrate (AEC 3-amino-9-ethylcarbazol)
A spot showing the place of the cytokine producer
cell
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It can be evaluated by microscopy (slow, manual
process) or you can use ELISPOT plate reader
(fast standardizable spot number and size
determination)
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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
Wehi 164 cell line
TNF is present
no TNF
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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
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.
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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
unknown cytokine containing solution
Disadvantage defined volume sample needed to
cover the surface of the membrane
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cytokine production of moDC activated by CD40L
and CD40LSLAM combination
Réthi és mtsi. 2006
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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)
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PCR
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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

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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.
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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
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(from Google pictures)
(Nature Protocols http//www.nature.com/nprot/jour
nal/v3/n6/images/nprot.2008.69-F1.jpg)
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Ficoll-Paque density based cell separation
pipettig the ring containing the mononuclear
cells to a new tube to get rid of ficoll
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Magnetic immunoseparation (MACS)
antigen specific antibody
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MACS
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Magnetic cell separation (MACS)
column
depleting or selecting unlabeled cells (negative
separation)
separation of labeled cells (positive separation)
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