methods.ppt

Techniques in immunology Techniques in immunology researchresearch

September 22 and 24, 2004

William Chan

ObjectivesObjectives

To recognize a number of techniques employed in To recognize a number of techniques employed in basic immunology research and be able to basic immunology research and be able to describe its principle.describe its principle.

Using a combination of the techniques discussed, Using a combination of the techniques discussed, to design an experimental approach to study an to design an experimental approach to study an immunology problem.immunology problem.

OverviewOverview

1.1. Flow cytometryFlow cytometry

2.2. MHC-peptide tetramersMHC-peptide tetramers

3.3. Magnetic cell sorting Magnetic cell sorting

4.4. ELISPOT assayELISPOT assay

5.5. Proliferation and killing assaysProliferation and killing assays

6.6. Adoptive transferAdoptive transfer

7.7. Animal modelsAnimal models

8.8. In vivo use of monoclonal antibodiesIn vivo use of monoclonal antibodies

References:

a) Immunobiology. 5th ed. Appendix I.

b) Kuby Immunology. 4th ed. Chapter 23.

Flow cytometryFlow cytometry

A technique that helps to characterize and enumerate A technique that helps to characterize and enumerate various cell types based on size, granularity and various cell types based on size, granularity and fluorescence intensity (using fluorescently labeled fluorescence intensity (using fluorescently labeled antibodies specific to cell surface markers)antibodies specific to cell surface markers)

Sorting of different cell populations is known as FACS Sorting of different cell populations is known as FACS (fluorescence-activated cell sorting).(fluorescence-activated cell sorting).

Fluidics

Optics

Electronics

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FACS.html

Forward and side scatterForward and side scatter

070503.001

0 200 400 600 800 1000FSC-HeightMouse blood

Granulocytes

LymphocytesCellular debris

Examples of applicationsExamples of applications

1.1. Determine the percentage of cells expressing a particular surface Determine the percentage of cells expressing a particular surface molecule.molecule.

2.2. Characterizing cell state.Characterizing cell state.

081803.006

100 101 102 103 104

TCR-FITC

R13

081803.006

100 101 102 103 104

CD4-TC

M1

Marker Events % Gated

All 4587 100.00

M1 4545 99.08

100 101 102 103 104

CD44-PE

Naïve Primed

MHC-peptide tetramersMHC-peptide tetramers

Four MHC-peptide complexes are associated into one molecule Four MHC-peptide complexes are associated into one molecule carrying a fluorescent labelcarrying a fluorescent label

Used to identify and quantitate Used to identify and quantitate antigen-specificantigen-specific T cells by flow T cells by flow cytometrycytometry

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0 200 400 600 800 1000FSC-Height

R1

072803.007

100 101 102 103 104

H-Y/Db-PE

Tetramer-specific T cells

Transgenic mouse containing a monoclonal population of CD8 T cells that recognize an antigen associated with class I MHC.

Magnetic cell sortingMagnetic cell sorting

A technique of cell separation by using magnetically labeled A technique of cell separation by using magnetically labeled microbeads against specific cell surface antigens.microbeads against specific cell surface antigens.

The magnetic microbeads are about 1,000,000 times smaller in volume than an eukaryotic cell and comparable to the size of a virus.

Type of selectionType of selection

PositivePositive Cells of interest are labeled and retained in the magnetic column Cells of interest are labeled and retained in the magnetic column

and then eluted as a purified fraction.and then eluted as a purified fraction.

Negative (depletion)Negative (depletion) Cells of interest are allowed to pass through column while unwanted Cells of interest are allowed to pass through column while unwanted

cells are magnetically labeled and retained in the column.cells are magnetically labeled and retained in the column. Preferred method of selection (why?).Preferred method of selection (why?).

ELISPOT assayELISPOT assay

Modified ELISA for measuring frequency of T cell Modified ELISA for measuring frequency of T cell responses.responses.

Used to determine what cytokine is produced following T Used to determine what cytokine is produced following T cell activation and what percentage of T cells are cell activation and what percentage of T cells are producing it.producing it.

T cells are activated by antigen of interest.

# of colored spots ÷ # of T cells added in assay = frequency of responding T cells

Functional studiesFunctional studies

Spleen cells from one source are cultured with spleen cells from a Spleen cells from one source are cultured with spleen cells from a different source to see whether a measurable response can be different source to see whether a measurable response can be detected and quantified.detected and quantified.

Response measured includes cellular proliferation (MLR) and killing Response measured includes cellular proliferation (MLR) and killing of target cells by cytotoxic effector cells (CTL assay).of target cells by cytotoxic effector cells (CTL assay).

MLRMLR

Mixed lymphocyte Mixed lymphocyte reaction.reaction.

Assess the Assess the responsiveness of T responsiveness of T cells (cellular cells (cellular proliferation following proliferation following activation) from one activation) from one source towards source towards antigens presented by antigens presented by cells from a second cells from a second source.source.

or spleen

*Important that stimulator cells are -irradiated (to induce DNA damage) prior to culture with responder cells

MLR(Female Marilyn responders at

5x104 cells)

0.0 0.5 1.0 1.5100

1000

10000

100000

Male Bm12Female B6Male B6

Stimulators (x106 cells)

CP

M

CTL assayCTL assay

5151Cr-release assayCr-release assay Assess the ability of CD8+ T cells stimulated with antigen to Assess the ability of CD8+ T cells stimulated with antigen to

generate effectors that kill radiolabeled target cells expressing the generate effectors that kill radiolabeled target cells expressing the same antigensame antigen

BALB/c responders(primed in vivo with B10.D2 spleen cells)

0.0 0.5 1.00

25

50

75

100

B6 targets

BALB/c targets

B10.D2 targets

Dilution of effectors

% ly

sis

Test target

Positive control

Negative control

Adoptive transferAdoptive transfer Transfer of immune cells from one host to another (with or without Transfer of immune cells from one host to another (with or without

prior irradiation of the recipient) to study an immune responseprior irradiation of the recipient) to study an immune response

T T

T

T T

T

Survives infection Succumbs to infection

Animal modelsAnimal models

In vivo systems (ie. use of animals to conduct research) In vivo systems (ie. use of animals to conduct research) are arguably better research tools than in vitro systems.are arguably better research tools than in vitro systems.

The mouse has been used extensively in immunological The mouse has been used extensively in immunological researchresearch

Well characterized genetically Well characterized genetically Easy to handle Easy to handle AffordableAffordable Rapid generation of progeny with a considerable litter sizeRapid generation of progeny with a considerable litter size Shares many similarities with the human immune systemShares many similarities with the human immune system

Transgenic miceTransgenic mice Mice carrying one or more transgenes (transgene = a cloned Mice carrying one or more transgenes (transgene = a cloned

foreign gene inserted into a living host)foreign gene inserted into a living host)

Gene knockout miceGene knockout mice

Specific genes can be deleted from the genome of a Specific genes can be deleted from the genome of a mouse in order to study the immunological functions of mouse in order to study the immunological functions of those genes.those genes.

Not all genes can be singularly deleted (ie. if they are embryonically Not all genes can be singularly deleted (ie. if they are embryonically lethal)lethal)

Absence of certain genes can significantly alter the immunological Absence of certain genes can significantly alter the immunological properties of the hostproperties of the host

Can knock out:Can knock out: Cells Cells Molecules expressed by cells (receptors, cell signaling proteins)Molecules expressed by cells (receptors, cell signaling proteins) Lymphoid tissuesLymphoid tissues Cellular products (cytokines)Cellular products (cytokines)

In vivo antibody treatmentIn vivo antibody treatment

Monoclonal antibodies (complete or only the F(ab’)Monoclonal antibodies (complete or only the F(ab’)22

fragment) against various antigens can be administered fragment) against various antigens can be administered into animals in order to observe the immunological into animals in order to observe the immunological effect(s) following antibody treatment.effect(s) following antibody treatment.

Functions of antibodies:Functions of antibodies: Deplete specific cell typesDeplete specific cell types Block or trigger cellular signals (e.g. costimulation)Block or trigger cellular signals (e.g. costimulation)

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100 101 102 103 104

CD3e-PE-Cy5

Quad Events % Gated

UL 905 15.87

UR 22 0.39

LL 1573 27.59

LR 3202 56.16

Quad Events % Gated

UL 36 0.46

UR 3 0.04

LL 445 5.66

LR 7383 93.85

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100 101 102 103 104

TCR-FITC

Baseline

7 days post-injection of NK-depleting antibody

SummarySummary

Many different research techniques are available for use Many different research techniques are available for use to help study various aspects of immunology.to help study various aspects of immunology.

More than one experimental approach can be taken to More than one experimental approach can be taken to study the same immunological problem.study the same immunological problem.