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Techniques in immunology research

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Title: Techniques in immunology research


1
Techniques in immunology research
  • September 22 and 24, 2004
  • William Chan

2
Objectives
  • To recognize a number of techniques employed in
    basic immunology research and be able to describe
    its principle.
  • Using a combination of the techniques discussed,
    to design an experimental approach to study an
    immunology problem.

3
Overview
  • Flow cytometry
  • MHC-peptide tetramers
  • Magnetic cell sorting
  • ELISPOT assay
  • Proliferation and killing assays
  • Adoptive transfer
  • Animal models
  • In vivo use of monoclonal antibodies
  • References
  • Immunobiology. 5th ed. Appendix I.
  • Kuby Immunology. 4th ed. Chapter 23.

4
Flow cytometry
  • A technique that helps to characterize and
    enumerate various cell types based on size,
    granularity and fluorescence intensity (using
    fluorescently labeled antibodies specific to cell
    surface markers)
  • Sorting of different cell populations is known as
    FACS (fluorescence-activated cell sorting).

5
Fluidics
Optics
  • Electronics

6
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/F/FACS.html
7
Forward and side scatter
Granulocytes
Cellular debris
Lymphocytes
Mouse blood
8
(No Transcript)
9
Examples of applications
  • Determine the percentage of cells expressing a
    particular surface molecule.
  • Characterizing cell state.

Naïve Primed
10
MHC-peptide tetramers
  • Four MHC-peptide complexes are associated into
    one molecule carrying a fluorescent label
  • Used to identify and quantitate antigen-specific
    T cells by flow cytometry

11
Tetramer-specific T cells
Transgenic mouse containing a monoclonal
population of CD8 T cells that recognize an
antigen associated with class I MHC.
12
Magnetic cell sorting
  • A technique of cell separation by using
    magnetically labeled microbeads against specific
    cell surface antigens.

13
The magnetic microbeads are about 1,000,000 times
smaller in volume than an eukaryotic cell and
comparable to the size of a virus.
14
Type of selection
  • Positive
  • Cells of interest are labeled and retained in the
    magnetic column and then eluted as a purified
    fraction.
  • Negative (depletion)
  • Cells of interest are allowed to pass through
    column while unwanted cells are magnetically
    labeled and retained in the column.
  • Preferred method of selection (why?).

15
ELISPOT assay
  • Modified ELISA for measuring frequency of T cell
    responses.
  • Used to determine what cytokine is produced
    following T cell activation and what percentage
    of T cells are producing it.

16
T cells are activated by antigen of interest.
of colored spots of T cells added in assay
frequency of responding T cells
17
Functional studies
  • Spleen cells from one source are cultured with
    spleen cells from a different source to see
    whether a measurable response can be detected and
    quantified.
  • Response measured includes cellular proliferation
    (MLR) and killing of target cells by cytotoxic
    effector cells (CTL assay).

18
MLR
  • Mixed lymphocyte reaction.
  • Assess the responsiveness of T cells (cellular
    proliferation following activation) from one
    source towards antigens presented by cells from a
    second source.

or spleen
19
Important that stimulator cells are g-irradiated
(to induce DNA damage) prior to culture with
responder cells
20
CTL assay
  • 51Cr-release assay
  • Assess the ability of CD8 T cells stimulated
    with antigen to generate effectors that kill
    radiolabeled target cells expressing the same
    antigen

21
Test target
Positive control
Negative control
22
Adoptive transfer
  • Transfer of immune cells from one host to another
    (with or without prior irradiation of the
    recipient) to study an immune response

T
T
T
T
T
T
Survives infection Succumbs to infection
23
Animal models
  • In vivo systems (ie. use of animals to conduct
    research) are arguably better research tools than
    in vitro systems.
  • The mouse has been used extensively in
    immunological research
  • Well characterized genetically
  • Easy to handle
  • Affordable
  • Rapid generation of progeny with a considerable
    litter size
  • Shares many similarities with the human immune
    system

24
Transgenic mice
  • Mice carrying one or more transgenes (transgene
    a cloned foreign gene inserted into a living host)

25
Gene knockout mice
  • Specific genes can be deleted from the genome of
    a mouse in order to study the immunological
    functions of those genes.
  • Not all genes can be singularly deleted (ie. if
    they are embryonically lethal)
  • Absence of certain genes can significantly alter
    the immunological properties of the host
  • Can knock out
  • Cells
  • Molecules expressed by cells (receptors, cell
    signaling proteins)
  • Lymphoid tissues
  • Cellular products (cytokines)

26
In vivo antibody treatment
  • Monoclonal antibodies (complete or only the
    F(ab)2 fragment) against various antigens can be
    administered into animals in order to observe the
    immunological effect(s) following antibody
    treatment.
  • Functions of antibodies
  • Deplete specific cell types
  • Block or trigger cellular signals (e.g.
    costimulation)

27
Baseline 7 days post-injection of
NK-depleting antibody
28
Summary
  • Many different research techniques are available
    for use to help study various aspects of
    immunology.
  • More than one experimental approach can be taken
    to study the same immunological problem.
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