context, cell mediated immunity refers to the induction of a potent
CD8
+ T cell response that can indirectly kill pathogens by targeting
infected or transformed cells. The activation of CD8
+ T cells can
similarly be assessed by flow cytometry using fluorochromeconjugated antibodies, while antigen-specific CD8
+ T cells can be
assessed using tetramer technology [5]. However, these techniques
do not inform on the cytotoxic abilities of a CD8
+ T cell to kill its
target in its host. CD8
+ T cell cytotoxicity assays were initially
developed as in vitro assays to assess ex vivo splenocytes (containing
activated CD8
+ T cells) for their ability to kill target cells (coated
with peptide antigen and
51
Cr or CFSE) in a controlled cell culture
system.
While this assay is highly useful for tracking CD8
+ T cell cytotoxicity over time as well as assessing the potency of the cytotoxic
CD8
+ T cell (CTL) response by varying the ratio of splenocytes to
target cells, a limitation of this assay is that the CD8
+ T cells are
required to kill their target in an artificial environment (culture dish
or well) and not in the host where other soluble or cellular factors
might be available that could modulate the response. Assessing
CTL cytotoxicity in vivo by intravenously injecting target cells
allows for the detection of CTL killing in their natural environment
in the whole organism [6].
To assess in vivo cytotoxicity in the mouse model, it is important to have a well-characterized antigen with a known CD8
+ T cell
epitope. To generate target cells capable of being recognized by the
CD8
+ T cell TCR, donor splenocytes must be incubated with
antigenic peptides that have a good affinity for the MHC class I
complex. We have used the model antigen ovalbumin protein for
immunization containing the well-characterized CD8
+ T cell epitope, SIINFEKL. A good adjuvant is also required to induce strong
CTL responses. We have used archaeosomes composed of a single
sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 60 -sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol)
that
when
admixed or used to encapsulate antigen results in the induction of
strong, long-lasting cellular and humoral immune responses [7];
however, the method described is applicable to other adjuvants.
Herein, a method is described to assess CTL killing in vivo by using
target cells coated with SIINFEKL peptide and labelled with a
fluorescent cytoplasmic dye CFSE.
1.1 Example
Experimental Overview
1. C57BL/6NCrl (C57BL/6) mice are immunized intramuscularly or subcutaneously with a vaccine formulation containing
20 μg ovalbumin and 1 mg SLA (see Note 1).
2. At various time-points after immunization, peptide-pulsed
CFSE-stained syngeneic donor splenocytes, known as “Target”
cells (Fig. 1), are injected intravenously into immunized
C57BL/6 mice (see Note 2).
550
Felicity C. Stark et al.
CD8
+ T cell response that can indirectly kill pathogens by targeting
infected or transformed cells. The activation of CD8
+ T cells can
similarly be assessed by flow cytometry using fluorochromeconjugated antibodies, while antigen-specific CD8
+ T cells can be
assessed using tetramer technology [5]. However, these techniques
do not inform on the cytotoxic abilities of a CD8
+ T cell to kill its
target in its host. CD8
+ T cell cytotoxicity assays were initially
developed as in vitro assays to assess ex vivo splenocytes (containing
activated CD8
+ T cells) for their ability to kill target cells (coated
with peptide antigen and
51
Cr or CFSE) in a controlled cell culture
system.
While this assay is highly useful for tracking CD8
+ T cell cytotoxicity over time as well as assessing the potency of the cytotoxic
CD8
+ T cell (CTL) response by varying the ratio of splenocytes to
target cells, a limitation of this assay is that the CD8
+ T cells are
required to kill their target in an artificial environment (culture dish
or well) and not in the host where other soluble or cellular factors
might be available that could modulate the response. Assessing
CTL cytotoxicity in vivo by intravenously injecting target cells
allows for the detection of CTL killing in their natural environment
in the whole organism [6].
To assess in vivo cytotoxicity in the mouse model, it is important to have a well-characterized antigen with a known CD8
+ T cell
epitope. To generate target cells capable of being recognized by the
CD8
+ T cell TCR, donor splenocytes must be incubated with
antigenic peptides that have a good affinity for the MHC class I
complex. We have used the model antigen ovalbumin protein for
immunization containing the well-characterized CD8
+ T cell epitope, SIINFEKL. A good adjuvant is also required to induce strong
CTL responses. We have used archaeosomes composed of a single
sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 60 -sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol)
that
when
admixed or used to encapsulate antigen results in the induction of
strong, long-lasting cellular and humoral immune responses [7];
however, the method described is applicable to other adjuvants.
Herein, a method is described to assess CTL killing in vivo by using
target cells coated with SIINFEKL peptide and labelled with a
fluorescent cytoplasmic dye CFSE.
1.1 Example
Experimental Overview
1. C57BL/6NCrl (C57BL/6) mice are immunized intramuscularly or subcutaneously with a vaccine formulation containing
20 μg ovalbumin and 1 mg SLA (see Note 1).
2. At various time-points after immunization, peptide-pulsed
CFSE-stained syngeneic donor splenocytes, known as “Target”
cells (Fig. 1), are injected intravenously into immunized
C57BL/6 mice (see Note 2).
550
Felicity C. Stark et al.