Lymphocyte subpopulation separation

This paper reviews the present status of affinity separation of cells based on the biospecific interaction of cellular receptors with proteinaceous ligands immobilized on a solid-phase matrix. Special emphasis was placed on the development of new matrix materials for immuno-affinity chromatography of lymphocyte subpopulations. Our newly developed matrix of poly(2-hydroxyethyl methacrylate)/polyamine graft copolymer offered novel advantages in (1) elimination of non-specific adsorption of lymphocytes and (2) simple immobilization procedure of ligand protein through non-covalent adsorption. This matrix allowed a rapid separation of preparative quantities of pure and vital lymphocyte subpopulations (IgG-positive and -negative cells) in excellent yield. 

Recently, affinity selection methods have also been shown to allow efficient separation of viruses, bacteria, cellular organelles, and even whole cells in their vital form (4-7). These methods offer a special advantage in the separation of lymphocyte subpopulations based on the biospecific interaction of immobilized ligands with marker proteins specifically expressed on the plasma membrane surface of each subpopulation. As reviewed in many articles (6-13), the separation of lymphocyte subpopulations has become increasingly important in the diagnosis as well as in the therapy of immuno-diseases, in donor-recipient matching in transplantation, and in the... 

Because the affinity panning method requires no special instrument for separation, it has been widely carried out as a laboratory technique for cell separation, especially for positive selection of lymphocyte subpopulations. Nevertheless, there are a number of disadvantages to this method as follows. First, the quantity of cells applicable to the dish is limited, and thus the method is not suitable for large scale separations. Secondly, a relatively long time-period, ca. 60 min, is required to allow all of the receptor-bearing cells to be stably anchored on the dish. 

As the separation of lymphocyte subpopulations based on the expression of immunoglobulin (Ig) molecules on their plasma membrane surfaces is of considerable practical interest at the present time (13), we have applied polyamine graft copolymers as solid-phase matrices for the separation of IgG-positive (IgG" ") and -negative (IgG ) lymphocytes. The solid-phase matrix was prepared by coating graft copolymers on glass beads of 48 - 60 mesh by solvent evaporation techniques. 

This test can be performed with the total peripheral blood lymphocyte population or with individual lymphocyte subpopulations (B lymphocytes, T4 and Tg lymphocytes) isolated from the total human lymphocyte fraction by monoclonal antibody separation. The rate of lymphocyte proliferation under influence of mitogens is measured through determination of the rate of incorporation of H-thymidine into the DNA of the lymphocytes. 

During recent years it has been established that lymphocytes are comprised of many subpopulations, such as T and B, mature and immature cells, etc. Although various techniques for lymphocyte separation have been devised, based mainly on cell size, density, electrical charge and specific surface antigens, none of these is satisfactory. Studies carried out recently in our laboratory have demonstrated that differential binding of lectins to sugars on different cell subpopulations can serve as a basis for a simple and effective method for cell fractionation. 

Ex vivo therapeutic strategies may take different forms. Chronic lymphocytic leukemias have been treated for long periods of time by using cell separators to reduce the burden of lymphocytosis, and to permit red cell transfusion. Laser-directed cell sorters may be used to select appropriate subpopulations of lymphocytes, which are then transfected with an appropriate gene product ex vivo and returned to the patient, where these cells will hopefully target some diseased tissue such as widespread melanoma. Expense, availability of therapy and the duration and specificity of effect currently limit the widespread application of these approaches. 

T lymphocytes may be separated into two major functional subpopulations having either regulatory or effector function. Regulatory cells represent about 70% of all T lymphocytes in the peripheral circulation and are separated into helper cells and suppressor cells. The remaining T cells have an effector function and are known as cytotoxic cells. 

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