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.