Immunological Testing - Cytotoxic T Lymphocyte Assays


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Immunological Testing

Cytotoxic T Lymphocyte Assays

Cytotoxic T lymphocytes (CTL) function to destroy cells that express foreign antigens. CTL play an important role in the elimination of virally infected cells. The majority of virus-specific CTL are CD8+. In contrast to CD4+ helper cells, which recognize antigen presented in the context of class II major histocompatibility complex (MHC) molecules, CTL recognize antigens in the context of class I MHC molecules. The importance of this distinction is that most of the time antigens need to be produced inside the cell in order to be presented by class I molecules. By contrast, antigens presented by class II MHC molecules can be taken up by antigen presenting cells, processed, and then presented to CD4+ cells.

The classical assay for CTL activity is the chromium release assay (73). Target cells expressing HIV antigen on their surface are labeled with a radioactive isotope of chromium (51Cr). Patient cells are then mixed with the target cell and incubated for several hours. Lysis of antigen-expressing cells releases 51Cr into the medium. HIV-specific lysis is calculated by comparing lysis of target cells expressing HIV or control antigens in the presence or absence of patient effector cells, and is usually expressed as the % HIV-specific lysis. Several studies have shown an inverse correlation between virus load and HIV-specific CTL activity, suggesting CTL play a role in controlling virus replication. This view is supported by data from SIV-infected macaques in which CD8 cells were transiently depleted, resulting in a prompt rebound in virus load.

Instead of measuring cytotoxicity, the CD8+ CTL response can be assessed by measuring IFN-a production by HIV-specific effector cells in an ELISPOT assay. In this assay, antigen-presenting cells (APC) are immobilized on the plastic surface of a microtiter well, and effector cells are added at various effector:target ratios. The binding of APC's by antigen-specific effector cells triggers the production of cytokines including IFN-a by the effector cells. The cells can be stained to detect the presence of intracellular IFN-a and the number of positively staining foci (spots) counted under a microscope. A second method for quantifying the number of circulating antigen-specific CD8+ T cells is the tetramer assay. In this assay, a specific epitope is bound to synthetic tetrameric forms of fluorescently labeled MCH Class I molecules. Since CD8+ T cells recognize antigen in the form of short peptides bound to Class I molecules, cells with the appropriate T cell receptor will bind to the labeled tetramers and can be quantified by flow cytometry. Although this method is less time-consuming than the ELISPOT assay, the tetramer assay measures only binding, not function. Not all cells that bind a particular antigen necessarily become activated. However, a recent paper demonstrated good correlation between ELISPOT, tetramer, and cytotoxicity assays (74). Another limitation of the tetratmer assay is that tetramers of all Class I subtypes are not available, so that

Lastly, CD8+ T cells also produce a soluble factor that capable of blocking HIV-1 infection, known as CD8 antiviral factor (CAF) (75). The precise nature of this factor is not known, and it might represent a mixture of several molecules. Production of this factor is triggered upon binding of specific antigens to CD8+ cells via the T cell receptor, but can also be triggered non-specifically. Production of the factor is assayed by testing dilutions of culture supernatant from appropriately stimulated cells for the ability to inhibit HIV replication in vitro. Chemokines are elaborated by stimulated CD8+ cells, and share certain similarities with CAF, but studies indicate that CAF is distinct from RANTES, MIP-1a, and MIP-1b.

Although the various assays for assessing virus-specific CTL have improved over the last five years, they are performed only in research laboratories and are not validated for clinical use. However, study of HIV-specific CTLs in various stages of disease provides important insights into AIDS pathogenesis and ultimately may lead to development of effective vaccine strategies.

4/15/01