T-cell mediated responses and T-cell function assays

Based on the current understanding of the immune response to cancerous cells, gliomas create a tumor microenvironment that suppresses immune function and allows the tumor to evade the host, cell-mediated, killing pathways. In the past, cellular immune responses were detected by measuring cytotoxicity, proliferation, or the release of cellular mediators such as cytokines. These laboratory tests typically involved in vitro preincubation of antigens with cultured cells. These analyses, however, were limited in their ability to estimate the quantity of cells that existed in a given population. The advent of the enzyme-linked immunosorbent spot (ELISPOT), Tetramer, cytokine flow cytometry, and chromium-release assays provide the capability of measuring clonal expansion of populations of antigen-responsive T cells.

A study by Galon and colleagues demonstrated that T-cell presence in colorectal cancer tissue is predictive of overall prognosis. Furthermore, data has demonstrated that T-regulatory cell activity increases in patients with known malignant gliomas. These cells are thought to later suppress cytotoxic T-cell activity and reduce the systemic response to tumor burden. Current clinical trials are monitoring systemic T-cell responses after vaccinations.

Enzyme-linked immunosorbent assay

Proteins that are secreted into the extracellular space, by tumor cells or neighboring host immune cells, can be monitored to identify multiple variables. These agents can identify if the host is amounting an attack on the tumor cells. It can also identify if the tumor is actively evading immune surveillance. Therefore, understanding the composition of circulating proteins in the intercellular space of tumor and self tissue provides valuable therapeutic and prognostic implications. The enzyme-linked immunosorbent assay (ELISA) was first developed in 1983 by Czerinkinsky and colleagues for the purpose of identifying and quantifying antibody secreting cells. It was later also used for quantifying antigens. The assay involves the administration of a known antibody to a solution of unquantified antibody or antigen. The known antibody is tagged with a known signal that is used for analysis and identification. It is common practice to add a second antibody to bind to the first administered antibody, to amplify the signal that is produced.

ELISAs are highly sensitive, reliable, and accurate tests. This method of analysis will prove invaluable in different methods of monitoring the immune response for various different therapies due to its accuracy and wide applicability in protein analysis.

Enzyme-linked immunosorbent assay

Proteins that are secreted into the extracellular space, by tumor cells or neighboring host immune cells, can be monitored to identify multiple variables. These agents can identify if the host is amounting an attack on the tumor cells. It can also identify if the tumor is actively evading immune surveillance. Therefore, understanding the composition of circulating proteins in the intercellular space of tumor and self tissue provides valuable therapeutic and prognostic implications. The enzyme-linked immunosorbent assay (ELISA) was first developed in 1983 by Czerinkinsky and colleagues for the purpose of identifying and quantifying antibody secreting cells. It was later also used for quantifying antigens. The assay involves the administration of a known antibody to a solution of unquantified antibody or antigen. The known antibody is tagged with a known signal that is used for analysis and identification. It is common practice to add a second antibody to bind to the first administered antibody, to amplify the signal that is produced.

ELISAs are highly sensitive, reliable, and accurate tests. This method of analysis will prove invaluable in different methods of monitoring the immune response for various different therapies due to its accuracy and wide applicability in protein analysis.

ELISPOT

The ELISPOT assay is a modified version of the ELISA immunoassay. ELISPOT assays were originally developed to quantify B cells secreting antigen-specific antibodies. They were later used to quantify the agents that were secreted by these cells, such as cytokines. The ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information. The ELISPOT assay is exceptionally sensitive, capable of detecting cells that secrete 100 molecules of a given protein. This exceptional sensitivity is because the protein of interest is rapidly captured around the secreting cell, before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. There is growing interest in increased use of ELISPOT as a measure for cytotoxic T lymphocytes (CTL) responses, in large part because it is reliable and highly sensitive. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, are now available and show the suitability of the ELISPOT assay for monitoring T-cell responses. The ELISPOT technique allows for quantification of tumor-specific T lymphocytes from peripheral blood by detecting antigen-induced cytokine secretion.

Tetramer analysis

A tetramer assay is used to detect the presence of antigen-specific T cells. In order for a T cell to detect the peptide to which it is specific, it must both recognize the peptide and the major histocompatibility complex (MHC) at the surface of a cell as it contacts it. Because the binding affinity of a T-cell receptor (TCR) to MHC complexed with a peptide is so low, creating a sensitive assay was historically difficult. This problem was solved by creating a tetramer of MHC molecules in which all four molecules present an identical peptide. In this manner, the avidity of the binding of the T cell of interest was increased. The resulting mechanism of the assay involves adding the MHC-peptide tetramer to an unknown quantity of T cells. The tetramer is then bound to the T cells that recognize the peptide sequence of interest. The tetramer is then stained typically by fluorescence labeling and bound T cells are measured using flow-cytometry.

Currently tetramer analysis has been used successfully in several phase I and II clinical trials for advanced staged melanoma vaccine trials and malignant gliomas. When combined with functional analyses such as staining for specific cytokines, tetramer analysis can provide valuable information about T cells and their activation state.