Proliferation

Lymphoproliferative assay (LPA): largely measures the in vitro proliferative capacity of CD4T cells. Typically PBMC are placed in culture with recombinant protein antigens for 5-7 days and then pulsed with 3H-thymidine to detect proliferation. It is dependent on (1) antigen processing and presentation; (2) the ability of T cells to proliferate in culture; and (3) the initial fequency of antigen-specific T cells.

Single cell Flow-cytometric methods: have recently be used to measure proliferative T cell responses to various stimuli including antigen. These procedures include intracellular markers of cell division including Ki67 in vitro or in vivo incorporation of BrdU, a thymidine analog, into DNA which can be detected by antibody and vital membrane dyes, for example, CFSE, which can be employed to determine the number of division cycles.

Assessment of CD8 T cell response

CTL Assay: Just as prolifreation has been employed as a measure of CD4T cell function, the cytotoxic T lymphocyte (CTL) assay has been a traditional measurement of the CD8 T cell resposne. The methods used to asses CTL function typically rely on long term culture of PBMC with antigen followed by incubation of activated T cells with 51 chromium-labeled MHC-matched target cells expressing the presented peptide or peptides of interest. Activity is typically based on the ratio of effector to target cells which promotes an arbitrary percentage of cell lysis (e.g., 50%) relative to a positive control for 51chromium release. The procedure is time consuming, uses large amounts of biological sample and because it measures population responses is poorly quantitative.

A potentially more valuable assessment of the CD8 T cell response is to measure the frequency of CTL precursor frequencies, that is, antigen-specific T cells capable of killing target cells expressing cognate antigen. In this respect, several markers have been associated with cytotoxic activity including granzymes, perforin, interferon-y and TNFalpha. CFV has also been used to correlate the frequency of cytomegalovirus (CMV) peptide induced CD8+ T cells expressing IFNy and/or perforin with CTL activity as measured by traditional 51Cr-release assays. Strong positive correlation has also been observed between specific lysis of peptide pulsed targets in a 51Cr-release assay and frequencies of peptide activated CD8+ T cells expressing IFNy at 6 or 6 days.

MHC oligomers: The binding of MHC class I restrcited tetramer complexes to cognate epitope specific T cells ahs been suggested as an anlternative method to identify CTL precursor frequencies. In comparative studies, the frquencies of cytokine psotive cells obtained by  have been similar to those obtained by MHC teramer analysis.

ELISPOT: An alternatie widely used assay for the determination of frequencies of antigen specific T celll responses is the ELISPOT, or enzyme-linked immunospot assay, which measures cytokine secretion by PBMC in filter bottom wells using immunochemical detection. While this procedure allows for single cell quantiation and a functional readout (cytokine production), it has the diadvantage ofbeing a singel parameter (or at most dual parameter) analysis, without the ability to easily discriminate subset phenotypes such as CD4+ and CD8+ T cells. The advantage is that multiple samples can be rapidly screened for positive cytokine responses.

Cell-Culture Systems

Primary Lymphoid Cell Cultures: Primary lymphoid cells cultures can be obtained by isolating lymphocytes directly from blood or lymph or various lymphoid organs which are then grown in a chemically basal medium (containing saline, sugars, vitamins, etc). 

 have been used to identify various cytokines involved in the activation, growth and differentiation of various cells involved in the immune response. For example, early experiments showed that media conditioned by the growth of various lymphocytes or antigen presenting cells would support the growth of other lymphoid cells. Many of the cytokines that characterized various conditioned media have been identified. The soluble growth factors elaborated by  are called monokines and the ones elaborated by  are called lymphokines.

MT-2 cells are chronically infected with HTLV-I. These are naive CD4+ T cells which express various activation markers such as CD25 and CD2.

Sap-T1 cells are not infected with a known retrovirus.

Cloned Lymphoid Cell Lines: One disadvantage with primary lymphoid cell cultures is that they consist of a heterogeneous group of cells that can be propagated for only a limited time. “Cell lines” can be propagated indefinitely which enables immunologists to obtain large numbers of homogeneous cells in culture. Although lymphoid cell lines survive indefinitely, they show various abnormal growth properties and often have an abnormal number of chromosomes. Some of these cell lines are derived from spontaneously occurring tumors of lyumphocytes, macrophages and other cells involved in the immune response. Other cell lines are induced by transformation of normal lyumphoid cells with viruses such as Abelson’s murine luekemia virus (A-MLV), simian virus 40 (SV40), Epstein-Barr virus (EBV) and HTLV-1. 

In 1973 it was serendipitously discovered that T cells could be maintained in tissue culture for extended period by culturing normal lymphocytes with IL-2. 

Hybrid Lymphoid Cell Lines: Normal antigen primed B cells can be fused with cancerous plasma cells (myeloma cells) and T cells can be fused with cancerous T cell lymphomas to form what is called a “hybridoma.” The fusion is usually done with polyethylene glycol. This hybridoma will contain a single nucleous with chromosomes from each of the fused cells. The hybridoma continues to express the antibody genes of the normal B lymphocyte or genes of the normal T cells but acquires the immortal growth properties of the cancerous T lymphoma cell.