Cytotoxic T Lymphocyte
Assays
Lab. 11
1
Cytotoxic T cells
• Cytotoxic T cells or lymphocytes (CTL) are one of the types of cells
that develop from lymphoid stem cells.
• They are major component of the cellular mechanism by which an
immune response leads to the destruction of foreign or infected
tissue.
• T cells are antigen specific, and carry antigen specific receptors.
• T cells also carry receptors for major histocompatability complex
(MHC) proteins, and will only function in the presence of the relevant
antigen and the appropriate MHC protein.
• Cytotoxic T lymphocytes interact with a target cell carrying the
relevant antigen
– e.g. a virus protein fragment on the surface of a cell infected with a
particular virus
– and also carrying the appropriate MHC Class 1 protein (or human
leukocyte antigen (HLA) Class 1 molecule),
– and the CTL release cytotoxic enzymes which act to kill the target cell.
2
3
Cytotoxic T cells
• T lymphocytes play a central role in:
– host defense directed against infectious agents and certain
malignancies.
– play a role in transplant rejection and organ-specific autoimmune
disease, uncontrolled T cell immune responses can be
pathogenic.
• Despite significant progress, the cellular and molecular
mechanisms that mediate these disease states are only
partially understood.
• Thus, the development of methods to describe and
quantify T cell immunity in humans may provide novel
insights into our basic understanding of human disease.
• Such methodology will aid in developing strategies
directed at improving clinical outcomes in a wide range
of disorders.
4
T cell immune response
• T cell immune response determine whether and
how the response is effective, ineffective or
inappropriately pathogenic.
• Clearly, clonal size (the number of antigenspecific cells induced) is a core characteristic,
• as low frequency responses may be inadequate
to control certain infections and therapy might be
aimed at boosting the frequency.
• In contrast, high frequency pathogenic T cell
immune responses may mediate more severe
pathology and therapy should be directed at
inhibiting the function of these cells.
5
Specificity of antigen-reactive T cells
• By determining the fine specificity for antigen-reactive T
cells, this could provide
– specific targets for immunotherapy aimed at appropriately
manipulating the quality and quantity of the response,
– so as ultimately to improve clinical outcome.
• Independent of clonal size and specificity, the cytokine
secretion pattern, as well as the ability to mediate
alternate effector functions such as cytotoxicity, can have
a large impact on the outcome of T cell immunity
• While a certain type of cytokine-secreting phenotype
may protect against certain intracellular infections, a T
cell immune response of similar specificity and
frequency, but producing IL-4 or IL-5, may not be able to
control the same pathogen.
6
Techniques for cytotoxicity measurment
• Cell mediated cytotoxicity can be mediated through
antibody or occur without a need for antibodies (CTL).
• Several different ways of target cell labeling for the
assessment of cytotoxic activity have been used.
• The two most common techniques use 51Cr release from
51Cr-labeled targets or an enzymatic assay measuring
release of intracellular substances.
• All these methods are based on measuring cell death
judged from plasma membrane disintegration and the
consequent release of cytoplasm.
• DNA fragmentation, a common event occurring during
apoptosis, is also an early event in the cell death caused
by cytotoxic cells.
• The JAM test is a sensitive and easy test for DNA
fragmentation and cell death.
7
Techniques for cytotoxicity measurment
• Another technique for measuring
cytotoxicity is ELISPOT assay.
• This identifies T cells that not only
recognize their target, but react to it by
producing cytokines, such as interferon.
• It offers a measure of functionality other
than cytotoxicity.
8
51
Chromium (Cr) Release Assays
• The CTLs Assay is used to detect the
cytolytic activity of Ag-specific
lymphocytes.
• The classical assay for CTL activity is the
chromium release assay.
• CTL’s function by destroying cells those
express foreign antigens (Ag) such as
virally infected cells: typically mediated by
CD8+ T-lymphocytes.
9
51
Chromium (Cr) Release Assays
• Cytotoxicity is relatively easy to measure,
there are straightforward ways to measure
cell death.
• If lymphocytes are taken from a mouse (or
a person) that was previously infected with
a virus, and mixed with other lymphocyte
cells infected with the same virus, the
infected cells will be killed.
10
51
Chromium (Cr) Release Assays
• Target cells are stimulated with an appropriate
peptide to activate the cells
• Target cells expressing the epitope of interest
are labeled with 51Cr
• Cells are incubated with CTL effector cells
• As the CTL effector cells bind to the antigen
specific target, the cells are lyzed and the 51Cr is
released into the culture supernatant
• The cells are centrifuged and the level of 51Cr in
the supernatant is measured by liquid
scintillation.
11
Assay for cytotoxic T cell function: Chromium release
12
Applications
• The method has been shown to work well in detecting
human immunodeficiency virus (HIV) specific CTL, using
peptide fragments of HIV.
• It is also applicable to detection of CTL specific for other
viruses.
• There is evidence that HIV specific CTL activity in
patients infected with HIV varies with development of
acquired immunodeficiency syndrome (AIDS) or related
conditions:
– many healthy HIV seropositive patients have a vigorous anti-HIV
CTL response,
– but there is evidence that HIV specific CTL activity declines as
disease progresses.
• Measurement of HIV specific CTL in samples from
patients infected with HIV may thus provide useful
information in following disease progression.
13
Disadvantages
• The main drawback to using 51Cr release
assays is the involvement of radioactive
reagents which:
– requires specialist laboratory certification
– and specially trained users.
– In addition the protocol can only be carried
out on fresh cells
14
The ELISPOT Assay
• Versions of the enzyme-linked immunospot
assay (ELISPOT) have been used for ~20 years
to detect antibody-secreting B cells and more
recently, cytokine-secreting lymphocytes.
• Several technical advances, including
– development of synthetic membranes
– and computer-assisted image analysis hardware and
software,
• have improved the reliability and reproducibility
of the assay and have facilitated the data
analysis.
15
The ELISPOT Assay
• To detect cytokine-secreting
lymphocytes, commercially
available 96-well ELISPOT plates
that have a white synthetic
membrane as a floor are coated
with a primary antibody specific
for the cytokine to be detected.
• Responding lymphocytes, in most
clinical situations peripheral blood
lymphocytes (PBLs) or purified
peripheral blood T cells (or T cell
subsets), are added to the wells
at varying dilutions
16
Principle
• These assays take advantage of the relatively
high concentration of a given protein (such as a
cytokine) in the environment immediately
surrounding the protein-secreting cell.
• These cell products are captured and detected
using high-affinity antibodies.
• The ELISPOT assay utilizes two high-affinity
cytokine-specific antibodies directed against
different epitopes on the same cytokine
molecule:
– either two monoclonal antibodies
– or a combination of one monoclonal antibody and one
polyvalent antiserum.
17
Results
• ELISPOT generates spots
based on a colorimetric reaction
that detects the cytokine
secreted by a single cell.
• The spot represents a “footprint”
of the original cytokineproducing cell.
• Spots are permanent and can
be quantitated visually,
microscopically, or electronically.
18
Steps of ELISPOT assay
The ELISPOT assay involves five specific steps:
(1) coating a purified cytokine-specific antibody to
a nitrocellulose-backed microtiter plate;
(2) blocking the plate to prevent nonspecific
absorption of any other proteins;
(3) incubating the cytokine-secreting cells at
several different dilutions;
(4) adding a labeled second anti-cytokine antibody;
(5) and detecting the antibody-cytokine complex.
19
In vitro assays for production of cytokines: ELISPOT assays
20
ELISPOT Assay Principle
Add PBMC
Prepare PBMC and count
wash
Coat plate with anti-cytokine Ab
Wash out cells, add detector Ab
24 h
1h
Add Ag
15 min
Count on
microscope
or
Analyze on
automated reader
21
Wash, add substrate
Applications
• Universities, research centers, cancer centers,
pharmaceutical companies, biotechnology companies,
clinical research laboratories and government related
research centers all have the potential to use ELISPOT.
• ELISPOT assays can be used for the quantitative
analysis of
– T cell responses to infections,
– identification of T cell epitopes,
– and for monitoring immunogenicity in vaccine trials.
• Uses might include monitoring of clinical trials involving
vaccinations against HIV, cancer, hepatitis B and C, and
other auto-immune disorders;
• monitoring of disease status and specific immune status;
and other basic scientific research, such as
inflammation, cell biology, transplantation, vaccine
22
development,