Cytotoxic T Lymphocyte Killing Assays | ProImmune

Cytotoxic T Lymphocyte Killing Assays

CD8⁺ cytotoxic T lymphocytes (CTL) are important effector cells of the immune system. By destroying cells that express foreign antigens on their surface through MHC class I molecules they protect against viral infections and also against certain cancers.

CTL Killing Assays At ProImmune

ProImmune routinely carries out assays for T cell function, and as part of our Special Assay Service we can build on our expertise to develop precisely the assay you require.

The cytotoxic capacity of CTL can be measured using a number of assays, which are described below.

⁵¹Chromium (Cr) Release Assay	Granzyme B ELISpot
CD107a Assay	Caspase-3 Assay
Other Flow Cytometric Assays	Further Reading

⁵¹Chromium (Cr) Release Assay

The classical assay to detect CTL activity is the ⁵¹Cr release assay. Essentially this assay assesses the ability of CD8⁺ cytotoxic T cells to lyze target cells expressing an epitope of interest.

Target cells are pulsed with an appropriate peptide antigen
Peptide-pulsed target cells are labeled with ⁵¹Cr
Target cells are incubated with CTL effector cells, usually at a range of effector:target ratios
As the antigen -specific CTL effector cells bind to their target, the cells are lyzed and ⁵¹Cr is released into the culture supernatant
The cells are centrifuged and the level of ⁵¹Cr in the supernatant is measured by liquid scintillation.

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; cryo-preserved cells cannot be used. Spontaneous ⁵¹Cr release from target cells can be an issue, as can poor labelling and low sensitivity. Finally, measurement of target cell lysis by ⁵¹Cr release assay does not provide any mechanistic information on the fate of target cells, or phenotypic information on the effector CTL.

Granzyme B ELISpot

Granzyme B is a serine protease released by CD8⁺ cytotoxic T cells upon interaction with target cells. Granzyme B directly cleaves the pro-apoptotic molecules BID, caspase-3 and caspase-7, activating target cell apoptosis. Release of Granzyme B is a sensitive indicator of CTL activity (1) (2).

ELISpot plates are coated with Granzyme B capture antibody
Target cells are pulsed with an appropriate peptide
Target cells are incubated with CTL effector cells, usually at a range of effector:target ratios
As the CTL effector cells bind to the antigen specific target, they release Granzyme B, which is captured on the ELISpot plate
After washing and detection steps, spots corresponding to the locations of effector cells can be visualized.

The advantage of Granzyme B ELISpot is that the number of cytokine-secreting CTL is measured directly.

CD107a Assay

Another measure of CTL activity is cell surface mobilization of CD107a (lysosomal-associated membrane protein-1, LAMP-1). CD107a is usually found in vesicle membranes, but during CTL-target cell interaction it is mobilized to the cell surface. This can be quantitated by flow cytometry

CTL are activated by culture with antigen-pulsed target cells, as before
anti-CD107a antibody is included during the stimulation
flow cytometry analysis is used to measure CD107a upregulation.

CD107a moblization correlates well with cytotoxic activity of CD8⁺ T cells (3) (4). CD107a is also expressed on NK cells and can be used as a marker of cytotoxic activity for NK cells. An advantage of using CD107a staining is that other staining can be used concurrently to gain detailed information about the phenotype of the activated CTL. Intracellular cytokine staining can be used to detect cytokines produced by CD107a-positive cells, and surface staining with MHC Pentamers can confirm their epitope specificity.

Caspase-3 Assay

Caspase-3 (CASP3) is a cysteine-aspartic acid protease . Sequential activation of this family of proteins, which are cleaved from proenzymes to proteolytically active forms, mediates cell death by apoptosis. Caspase-3 is activated in target cells after encounter with CTL, so it can be used as a measure of CTL cytotoxicity (5) (6). Caspase-3 activation can be measured using a variety of reagents.

Using a synthetic substrate which changes colour upon cleavage
Using an antibody to the cleaved (activated) form of caspase-3
Using a fluorescently-conjugated, cell-permeable irreversible inhibitor of activated Caspase-3

The assay is more sensitive and simpler to perform than ⁵¹Cr release assays, and can be combined easily with staining for other markers, or the use of cell tracker dyes, to gain more information about the events taking place in the target cell population immediately following CTL encounter.

Flow Cytometric Assays

A very straightforward measurement of CTL activity can be made using a combination of fluorescent cell membrane dyes (eg. PKH-26) and DNA-intercalating dyes (eg. TO-PRO®-3 iodide), and flow cytometry, and is comparable to the ⁵¹Cr release assay (7).

Target cells are pulsed with an appropriate peptide antigen, and labelled with a membrane dye.
Target cells are incubated with CTL effector cells, usually at a range of effector:target ratios
As the antigen -specific CTL effector cells bind to their target, the cells are lyzed. A cell impermeant DNA-intercalating viability dye is added.
The samples are analysed by flow cytometry.

Using this method, dead target cells will appear as double-positive for TO-PRO®-3 iodide and PKH-26, and can be distinguished from live target cells (PKH-26 single-positive) and CTL (negative for both). Addition of Annexin-V staining can be used to distinguish early-apoptotic target cells - Annexin V stains phosphatidylserine, which is usually restricted to the inner leaflet of the plasma membrane but is externalized upon initiation of apoptosis.

References

(1) Shafer-Weaver K et al (2003) The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity. Journal of Translational Medicine 1:14. [PubMedID: 14697097]

(2) Rininsland F et al (2000) Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity. Journal of Immunological Methods. 240:143-155. [PubMedID:10854609]

(3) Betts MR et al (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation .Journal of Immunological Methods 281:65-78.[PubMedID:14580882]

(4) Aktas E et al (2009) Relationship between CD107a expression and cytotoxic activity. Cellular Immunology 254:149-54 [PubMedID:18835598]

(5)Jerome KR et al (2003) Measurement of CTL-induced cytotoxicity: The caspase 3 assay. Apoptosis, 8: 563-571 [PubMedID: 14574062]

(6) He L et al (2005) A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. .Journal of Immunological Methods 304: 43-59 [PubMedID: 16076473]

(7) Lee-MacAry AE et al (2001) Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. .Journal of Immunological Methods 252:83 [PubMedID:11334968]

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