A novel micro-cell-mediated lympholytic assay for the evaluation of regulatory cells in human alloreactive CTL responses.

Since cell-mediated lympholysis (CML), the most commonly used in vitro experimental cytotoxic method for the evaluation of regulatory cells, requires large numbers of cells that are often the limiting factor, we have developed a new micro-cell-mediated lympholytic (m-CML) assay. Various numbers of responding cells were stimulated with equivalent numbers of allogeneic irradiated stimulator cells in the presence of (fivefold) serial dilutions of regulatory cells. On the 8th day, 4-h 51Cr-release assays were performed by adding 5000 labeled target cells from the corresponding stimulators to the cultures. Even though results that were comparable to the macro- (bulk) CML and MLR modulation experiments were obtained with all the cell combinations tested in the m-CML, the combinations with 50,000 responder cells and stimulating cells and dilutions of 25,000 to 40 modulator (regulatory) cells were found to be the most reproducible for assaying regulatory cell potency in vitro. Similarly, expression of the results as percentage inhibition using percent specific lysis values was the simplest method of calculation. This assay was standardized for the evaluation of the inhibitory activity of a variety of regulatory cells, including long-term cultures of cadaver-donor vertebral body bone marrow cells (vDBMC-L), in vitro generated CD8 positive and CD28 negative suppressor T cells and donor chimeric cells isolated from renal transplant recipients who had been perioperatively infused with donor bone marrow cells (DBMC). The results indicate that the m-CML assay is a sensitive and reliable micromethod with at least 10-fold fewer responders, stimulator and modulator cell numbers needed than macro-CML assays for the evaluation of regulatory cells obtained from a variety of immune systems in vitro.