Measuring the functional activity of T and B lymphocytes

Polyclonal activation of T and B cellslectin-induced activationα-IgM, α-CD3 or α-TCR antibodyallogeneic T cell activation(examination of the immediate-early activation events)

T and B cell responseactivation markersproliferative response: 3H-thymidine incorportion

CFSE fluorescence decreasecell 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 stainingMHC tetramers

Phases of the humoral immune response(review)

Phases of T cell response(review)

Lymphocyte function can be investigated by polyclonal T/B-lymphocyte activator materials

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

Polyclonal activation of lymphocytes by LPS, lectins, PMA/ionomycin

A B C

BCR or TCR-specifc antibodies could activate the lymphocytes also

(PMA activates protein kinase C)

TLR4

B cell T cell T cell

Polyclonal B cell activators

In the presence of cytokinesyesAnti-Ig

yesyesEBV (transforming effect)

yesnoSpA (superantigen, staphylococcus protein A)

yesnoPWM (pokeweed mitogen)

Human B cells

Ig secretionT cell dependencyActivator

Polyclonal T cell activatorsPhytohaemagglutinin (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)ChenopodialesPhytolaccaceae

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(seconds-minutes)

- Western blot- Bead array

I.c Ca2+ increase - FACS, microscopy

Gene activation - RT-PCR

Cytokine synthesis

Cytokine secretion

- i.c cytometry

- ELISA, ELISPOT- Bioassay

Antigen receptors (TCR, BCR), and different other receptors (eg. cytokine receptors)

Cell-cycle/apoptosis - DNA content- IN antigens

Cell division - 3H-thymidine, CFSE, MTTLymphocyte activationThe examination often requires specific

Ag-Ab reactions

Western blottingSteps:

1. sample preparation (cells, tissues)2. gel electrophoresis3. blotting4. labeling5. development

use:

Identification of defined components from protein mixtures by antigen specific antibodies

Anode(+)

Cathode(-)

• 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

StandardProtein sample

SDS-PAGE Membrane Western blot

Antibody recognizes epitope in specific protein

Western Blot

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

Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by by Western blotWestern blot

X-linked agammaglobulinemia. XLA patients do not generate mature B cells, which manifests as an almost complete lack of antibodies in their bloodstream.

Futatani T et al. Blood 1998;91:595-602

Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by flow cytometryby flow cytometry

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

An increase in cytoplasmic Ca2+ levels can be detected by

fluorescent indicator dyes./Fluo-3 or Indo-1/

Detection of intracellular Ca2+ concentration

Measurement of Ca2+ signal by flow cytometry

Flu

ore

scen

ce p

rop

ort

ion

al

wit

h I

ntr

acel

lula

r C

a2+ l

evel

time

basic signal

activation of cells

e.g. Fluo-3or

Indo-1

You can detect by fluorimeter also

Measurement of Ca2+ signal by flow cytometryT cell hybridoma specific for influenza virus hemagglutinin protein-derived

peptide - Ca2+ signal by antigen presentation

non-activated T cells

T cell activation(APC - T cell)

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)

Tissue sample

Fixation

Section before staining

Freezing

Sectioning

Immunohistochemistry

Immunohistochemistry ABC Method

Secondary antibody

Avidin

Cells

Slide

Primary antibody

Biotin

Enzim

X

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 detectionDetection 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 cytometryFlow 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 Can investigate enormous number of cells in short period of timeof 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 CytometryExample Chanel Layout for Laser-based Flow Cytometry

PMT 1

PMT 2

PMT 4

The emited fluorescent light can be separeted to components by

special mirrors and filters

Laser

PMT 3cell

suspension in tube

photodetectors

flow cell

forward light scatter detector

(PMT=photo-multiplayer tube)

Laser

Light scatter and fluorescence

Forward angle light scatter

sensor(FSC, FALS)

Side light scatter(SSC) and fluorescence detectors

Can be loosely considered as a

representation of the particle size

represents the granularity of the cells

Multocolor staining can be used to identify cell sub-populations

* autofluorescence – presence of piridins and flavins.

BNKTh Tc

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)

Lymphocytes in the periferial blood

Fluorescent microscopy

Immunophenotyping

focu

sed

lase

r bea

m

high velocity flow stream(in cuvette or stream in air)

CD4FITC

CD

8P

E

de

tec

tor

detecting CD4-FITC labeled (TH) cell

signal processing unit

screen

a dot representing aCD4+ CD8- cell

increasing light intensity

microscopy:

de

tec

tor

detecting the PE labeled cell(CD8-PE)

CD

8P

ECD4FITC

signal processing unit

increasing light intensity

de

tec

tor

detecting the unlabeled cell(eg.B cell) by autofluorescence

CD

8P

ECD4FITC

Signal processing unit

increasing light intensity

microscopy: dim (autofluorescent)

cell

quadrantstatistics

CD

8P

ECD4FITC

38%

0%

44%

18%

Graphical representations 1.

dot-plot

contour-plot

density-plot

Graphical representations 2.

Histogramm

homogenous cell population is normally distributed (Gaussian)

Numeral intensity values:

~ 7 ~ 1300

Different cell types - Different cell types - characteristic light scatteringcharacteristic light scattering

forward light scattering (FSC)

(„size”)

side light scattering (SSC)(e.g. granulated)

granulocytes

monocytes

lymphocytes

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)

Only the major cell types can be identified

1 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils

Characterisation of immune cells using cell Characterisation of immune cells using cell surface markerssurface 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+ = 1.6Normal 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

WAS: Wiscott-Aldrich Syndrome

XLA: X-linked Agammaglobulinemia

Inhibited B cell development: Lack of CD19+ B cells

A typical symptom: Lacking or decreased CD43 expression

Diagnosis of immunodeficiency by flow cytometry

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)

cytokines

- optionally the cells could be labelled by some cell type specific

antibody in the beginning (e.g. CD4)

One can determine which cell type produced the cytokines!

ELISA

ELISA plate

Enzyme Linked Immune Sorbent Assay

well

enzyme linked immune sorbent

Antibody conjugated with enzyme

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

Direct method Indirect method

Antigen

Primary antibodies

Label

Label

Secondary antibodies

For antigens present at low concentration in complex biological samples

Removal of unbound material

Blocking free plastic surface with inert

protein

Removal of unbound protein

Addition of biotinylated antibody specific to a different epitope on

target protein

Removal of unbound material

Addition of avidin-conjugated enzyme

Addition of substrate

Coating with Ag-specific „capture”

antibody

Addition of antigen- containing solution

Removal of excess enzyme

Steps of combined sandwich ELISA

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

nitrocellulose membrane(signal detection pad)

glass fiber membrane with visually labeled detection antibodies

front view side view

hCG capture antibody lane

control antibody lane(detection antibody capture)

absorbtion pad (cellulose)

sample application pad

urine

hCG capture antibody line

control antibody line

detection antibodies

hCG

control line (C)

test line (T)

hCG + hCG negative

detection antibody capture antibodies

hCG line( bound hCG)

control linedetection antibody

capture antibodycontrol line

test line

hCG positive hCG negative

Competitive system

ELISA plates - resultsELISA plates - results

ELISPOTEnzyme Linked Immuno-Spot

The principles are similar to ELISACapable to determine the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector moleculesThe 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 activationThe first steps should be done in aseptic conditions

ELISPOT- coating with antigen specific capture antibodies

- blocking

- administration of the cells

- administration of biotin conjugated antigen specific secondary antibody

- avidin-enzyme conjugate

- administration of the unsoluble chromogenic substrate (AEC 3-amino-9-

ethylcarbazol)

(activation, incubation)

- washing

A spot showing the place of the cytokine

producer cell

Upper view of a well on an ELISPOT plate with the generated spots

The process

It can be evaluated by microscopy (slow, manual process) or you can use “ELISPOT plate reader” (fast + standardizable spot number and size determination)

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.

BioassayBioassay

Wehi 164 cell line

CTLL2 cell line

IL-2 is present no IL-2

TNF is present no TNF

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.

Living cells can be visualized by colorimetric assay (eg. MTT), or their proliferation can be measured by other methods.

cytokine concentration

IL-2

TNF

CTLL2CTLL2

Wehi 164Wehi 164

MTT assay living cells convert the stain to purple-blue

Cytokine arrayCytokine array(The process is similar to the procedures of Western-blot after the blotting step)

( - )

IFN

( + ) IL-2 IL-4 IL-5

…

IL-12 …

multiple antigen specific antibodies bound to membrane

unknown cytokine containing solution

„Luminescent antibody” mixture

Multiple cytokines can be detected rapidly in the same procedure

Disadvantage – defined volume sample needed to cover the surface of the membrane

cytokine production ofmoDC activated by CD40L andCD40L+SLAM combination

Réthi és mtsi. 2006

method: RT-PCR, QRT-PCR

Investigation of gene activationInvestigation 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

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

Characteristics of the separation:

• purity• recovery, yield, lost• viability of the cells

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!)

Cell separationCell 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.

SeparationSeparation

The different density parts of the anticoagulated blood has been separating to three parts in undisturbed tube:bottom: sedimented red blood cellstop: cell free plasmathe intermediate layer is called „buffy coat” contains the leukocytes, platelets

The process can be accelerated by centrifugation

(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)

(from Google pictures)

Ficoll-Paque: density based cell separationFicoll-Paque: density based cell separation

peripheral blood

pipetting cells on ficoll

centrifugation

separated cells

plasma

ficoll

Red blood cells

mononuclear cells

(PBMC)

neutrophilgranulocytes

pipettig the „ring” containing the

mononuclear cells to a new tube

to get rid of ficoll

Magnetic immunoseparation (MACS)

paramagneticbead

antigen specific antibody

MACS

Magnetic cell separation (MACS)

MA

GN

ETM

AG

NE

T

column

depleting or selecting unlabeled cells

(negative separation)

separation of labeled cells(positive separation)

Magnetic column