Enrichment and isolation of B lymphocytes

Depletion

The first attempts to deplete antigen-binding cells were made by Wigzell and colleagues using antigen-coated beads in affinity columns, which depleted antigen-binding cells but also a significant number of nonspecific cells. An alternative approach was to deplete antigen-specific cells by allowing them to bind radioactive antigen of high enough specific activity to kill them; a technique sometimes referred to as 'antigen suicide'. Ada and Byrt demonstrated that incubation in the cold of lymphoid cells with l25I-labeled T-independent antigens specifically abolished the response of these cells to the unlabeled antigen on adoptive transfer into irradiated syngeneic recipients, establishing that antigen-binding B cells were the precursors of antibody-forming cells. These experiments provided strong support for the clonal selection theory but did not yield purified populations of antigen-specific cells for further study. Such methods are no longer widely used.

Enrichment: historical methods

A number of approaches have been taken to enrich lymphocyte populations for antigen-specific cells. Some are now of historical interest only. Such techniques include the use of affinity matrices and columns. Two major problems with these modalities were nonspecific adsorption of cells to affinity matrices and difficulty in recovering viable, functional cells after interaction with antigen.

Another early method for isolating antigen-specific cells was the use of hapten-gelatin-coated Petri dishes ('panning'). Cell suspensions were added to the dishes, binding of hapten-specific B cells occurred, unbound cells were washed off and bound cells recovered by melting the gelatin layer at 37°C. A major disadvantage of this method was its relatively low cell yield, but combined with limiting dilution assays, it was a useful method for studying antigen-specific B cell activation.

Rosetting has also been used to enrich for antigen-specific B cells. For example, haptenated horse red blood cells (HRBCs) were allowed to bind to hapten-specific B cells, forming rosettes. The rosettes could then be separated from nonrosetted cells by buoyant density and/or sedimentation velocity centrifugation, and the HRBCs removed from the antigen-binding cells by protease digestion, followed by overnight culture of the cells to allow for receptor regeneration.

Enrichment: 'Pre-enrichment' by depletion of nonantigen-specific cells

Partial purification or enrichment of antigen-specific cells subsequently allows more rapid and accurate isolation of these cells by more time-consuming or expensive procedures, such as fluorescence-activated cell sorting (FACS: see below). The removal of unwanted cells can be achieved by physical means, such as by taking advantage of the propensity of T cells to bind to nylon wool columns or of macrophages to adhere to plastic. Depletion of unwanted cells using complement fixation is a commonly used and effective technique. To enrich for immunoglobulin G-positive (IgG+) B cells, for example, spleen cells are cultured in a cocktail of antibodies against

T cells (e.g. anti-CD3), macrophages and other B cells (e.g. anti-IgM). Provided the chosen antibodies fix complement efficiently (IgM antibodies are best) and have been properly titrated to reduce nonspecific killing, considerable enrichment and high yields can be achieved. Similar cocktails of antibodies conjugated directly or indirectly (via biotin-avidin) to magnetic beads can be used to remove unwanted cells, as cells binding the paramagnetic beads will stick to steel wool in a column or to the side of a container when a magnetic field is applied, while the desired cells will pass through the column (Figure 1). A potential hazard of this technique is the risk of saturating the column: if the surface area of the steel wool is inadequate to bind the number of cells to be depleted they too will pass through the column causing substantial contamination. One advantage of these 'negative selection' methods of enrichment is that they do not require antibody binding to, and thus potential activation of, the antigen-specific cells.

Enrichment: positive 'pre-enrichment'

Paramagnetic beads can also be used to enrich for antigen-specific B cells in a 'positive' fashion. If, for example, the antigen-specific B cells have a characteristic immunoglobulin isotype (such as the 7 heavy and \ light chains in the response to NP) antibodies to these can be used to attach magnetic beads to the cells of interest. These cells will then attach to the column as described above, and can be washed by passage of medium through the column, followed by their removal by flushing after withdrawing the column from the magnetic field (Figure 1). Saturation of the column will not result in impurity of the resulting population, but only a reduced yield. In the example above, enrichment of spleen cells using anti-IgGl and paramagnetic beads can result in a 40-fold enrichment for anti-NP-specific B cells, with a proportionate reduction in the flow cytometry time needed to complete the purification.

One problem with positive enrichment using these columns is nonspecific adhesion of dead cells to the column. If this is a problem, dead cells may have to be removed in a separate initial step, for example using density gradient separation. Direct conjugation of antigen to paramagnetic beads is currently being attempted which should improve isolation of antigen-specific cells by this method.

The flow cytometer can itself be used to pre-enrich cells prior to a further round of flow cytometry. Very rapid sorting by FACS results in a predictable number of errors and therefore impurity. Substantial enrichment is nonetheless achieved during this very rapid sorting, and the time needed for the subsequent

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