During the 1960s, early in the history of modern cellular immunology, it was observed that when rat lymphocytes were cultured on a monolayer of mouse fibroblast cells, the rat lymphocytes proliferated and destroyed the mouse fibro-blasts. In 1970 it was discovered that functional CTLs could also be generated by co-culturing allogeneic spleen cells in a system termed the mixed-lymphocyte reaction (MLR). The T lymphocytes in an MLR undergo extensive blast transformation and cell proliferation. The degree of proliferation can be assessed by adding [3H] thymidine to the culture medium and monitoring uptake of label into DNA in the course of repeated cell divisions.
Both populations of allogeneic T lymphocytes proliferate in an MLR unless one population is rendered unresponsive by treatment with mitomycin C or lethal x-irradiation (Figure 14-16). In the latter system, called a one-way MLR, the unresponsive population provides stimulator cells that express alloantigens foreign to the responder T cells. Within 24-48 h, the responder T cells begin dividing in response to the alloantigens of the stimulator cells, and by 72-96 h an expanding population of functional CTLs is generated. With this experimental system, functional CTLs can be generated entirely in vitro, after which their activity can be assessed with various effector assays.
The significant role of TH cells in the one-way MLR can be demonstrated by use of antibodies to the TH-cell membrane marker CD4. In a one-way MLR, responder TH cells recognize allogeneic class II MHC molecules on the stimulator cells and proliferate in response to these differences. Removal of the CD4+ Th cells from the responder population with anti-CD4 plus complement abolishes the MLR and prevents generation of CTLs. In addition to TH cells, accessory cells such as macrophages also are necessary for the MLR to proceed. When adherent cells (mostly macrophages) are removed from the stimulator population, the proliferative response in the MLR is abolished and functional CTLs are no longer generated. It is now known that the function of these macrophages is to activate the class II MHC-restricted TH cells, whose proliferation is measured in the MLR. In the absence of TH-cell activation, there is no proliferation.
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