malignant tumor growth [7–9]. ELISpot can also be used to guide
the proper application of viral gene therapy vectors by measuring
cellular responses to the viral proteins [10].
Lymphocytes are a type of white blood cell that play a central
role in the body’s immune defense and include T, B and natural
killer cells. T cells mediate the adaptive antigen-specific cellular
immune responses and are subdivided into many subtypes including CD4+ T helper and CD8+ cyotoxic T cells. CD8+ and CD4+ T
cells are specific to a particular epitope which they can bind in the
context of a major histocompatibility complex (MHC) on the
cellular surface [1]. Upon binding of their epitope, CD4+ and
CD8+ T cells are activated resulting in the release of cytokines
such as IFN-γ. Cytotoxic CD8+ T cells can also respond by releasing lytic granules with the aim of killing the undesired host cell
which has been deemed to be a risk due to its presentation of a
pathogen or cancer-specific epitope. While CD4+ cells predominantly support the immune response by producing and secreting
cytokines, they can also have lytic activity [11]. In studies evaluating
vaccine activity, the use of ELISpot to quantify antigen-specific T
cells relies most commonly on detecting IFN-γ expression due to its
(1) strong correlation with CD8 T cell lytic activity and (2) production in CD4 T cells involved in the activation of cytotoxic immune
responses [3, 12]. Herein, we describe an ELISpot-based method
to measure ovalbumin-specific CD4+ and CD8+ T cells in mouse
samples (Fig. 1). Ovalbumin is routinely used as a model antigen
when evaluating vaccine adjuvants in preclinical studies as (1) it is
an inherently weak immunogen when administered alone and
(2) the epitopes presented by specific laboratory mouse strains are
well characterized. In a sandwich ELISA, the analyte of interest is
already present in the test sample prior to addition to the ELISA
plate to be bound by the capture and detection antibodies. With
ELISpot, cells are added directly to the plate and stimulated with
the antigen of interest to generate the test analyte after their addition to the plate. The capture antibodies on the well surface directly
adjacent to the stimulated cell will bind the cytokine in real time as
it is being produced. Roundish spots will form once the plates are
developed, and based on the one spot–one cell principle, we can
estimate the number of cells producing an analyte such as IFN-γ
[13]. This method has allowed for the determination of the capability of a vaccine formulation to induce antigen-specific T cells and
also study the kinetics and longevity of these cellular responses. It
can be easily adapted to different antigens by replacing the stimulant peptides.
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Bassel Akache and Michael J. McCluskie
the proper application of viral gene therapy vectors by measuring
cellular responses to the viral proteins [10].
Lymphocytes are a type of white blood cell that play a central
role in the body’s immune defense and include T, B and natural
killer cells. T cells mediate the adaptive antigen-specific cellular
immune responses and are subdivided into many subtypes including CD4+ T helper and CD8+ cyotoxic T cells. CD8+ and CD4+ T
cells are specific to a particular epitope which they can bind in the
context of a major histocompatibility complex (MHC) on the
cellular surface [1]. Upon binding of their epitope, CD4+ and
CD8+ T cells are activated resulting in the release of cytokines
such as IFN-γ. Cytotoxic CD8+ T cells can also respond by releasing lytic granules with the aim of killing the undesired host cell
which has been deemed to be a risk due to its presentation of a
pathogen or cancer-specific epitope. While CD4+ cells predominantly support the immune response by producing and secreting
cytokines, they can also have lytic activity [11]. In studies evaluating
vaccine activity, the use of ELISpot to quantify antigen-specific T
cells relies most commonly on detecting IFN-γ expression due to its
(1) strong correlation with CD8 T cell lytic activity and (2) production in CD4 T cells involved in the activation of cytotoxic immune
responses [3, 12]. Herein, we describe an ELISpot-based method
to measure ovalbumin-specific CD4+ and CD8+ T cells in mouse
samples (Fig. 1). Ovalbumin is routinely used as a model antigen
when evaluating vaccine adjuvants in preclinical studies as (1) it is
an inherently weak immunogen when administered alone and
(2) the epitopes presented by specific laboratory mouse strains are
well characterized. In a sandwich ELISA, the analyte of interest is
already present in the test sample prior to addition to the ELISA
plate to be bound by the capture and detection antibodies. With
ELISpot, cells are added directly to the plate and stimulated with
the antigen of interest to generate the test analyte after their addition to the plate. The capture antibodies on the well surface directly
adjacent to the stimulated cell will bind the cytokine in real time as
it is being produced. Roundish spots will form once the plates are
developed, and based on the one spot–one cell principle, we can
estimate the number of cells producing an analyte such as IFN-γ
[13]. This method has allowed for the determination of the capability of a vaccine formulation to induce antigen-specific T cells and
also study the kinetics and longevity of these cellular responses. It
can be easily adapted to different antigens by replacing the stimulant peptides.
526
Bassel Akache and Michael J. McCluskie