Although FACS is superb for isolating small numbers of cells in pure form, mechanical means of separating cells are preferable when large numbers of lymphocytes must be prepared quickly. A powerful and efficient way of isolating lymphocyte populations is to couple paramagnetic beads to monoclonal antibodies that recognize distinguishing cell-surface molecules. These antibody-coated beads are mixed with the cells to be separated and are run through a column containing material that attracts the paramagnetic beads when the column is placed in a strong magnetic field. Cells binding the magnetically labeled antibodies are retained; cells lacking the appropriate surface molecule can be washed away (Fig. A.22). The retained cells are recovered by removing the column from the magnetic field. In this case, the bound cells are positively selected for expression of the particular cell-surface molecule, while the unbound cells are negatively selected for its absence.
A-20 Isolation of homogeneous T-cell lines.
The analysis of specificity and effector function of T cells depends heavily on the study of monoclonal populations of T lymphocytes. These can be obtained in four distinct ways—T-cell hybrids, cloned T-cell lines, T-cell tumors, and
Fig. A.22 Lymphocyte subpopulations can be separated physically by using antibodies coupled to paramagnetic particles or beads. A mouse monoclonal antibody specific for a particular cell-surface molecule is coupled to paramagnetic particles or beads. It is mixed with a heterogeneous population of lymphocytes and poured over an iron wool mesh in a column. A magnetic field is applied so that the antibody-bound cells stick to the iron wool while cells that have not bound antibody are washed out; these cells are said to be negatively selected for lack of the molecule in question. The bound cells are released by removing the magnetic field; they are said to be positively selected for presence of the antigen recognized by the antibody.
limiting-dilution culture. By analogy with B-cell hybridomas (see Section A-7), normal T cells proliferating in response to specific antigen can be fused to malignant T-cell lymphoma lines to generate Tcell hybrids. The hybrids express the receptor of the normal T cell, but proliferate indefinitely owing to the cancerous state of the lymphoma parent. T-cell hybrids can be cloned to yield a population of cells all having the same T-cell receptor. When stimulated by their specific antigen, these cells release cytokines such as the T-cell growth factor interleukin-2 (IL-2), and the production of cytokines is used as an assay to assess the antigen specificity of the T-cell hybrid.