Mark Brunswick

Proliferative Assays for B Cell Function

This unit describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen‐induced proliferation) requires the laborious procedures of isolating antigen‐specific B cells (which are otherwise present in too low a concentration in whole B cell populations). Cross‐linking of the B cell antigen receptor, surface immunoglobulin (sIg), by specific antigen stimulates cells to proliferate prior to secreting Ig. For this purpose, monoclonal or heterologous affinity‐purified anti‐Ig antibodies are used. B cells can also be stimulated to proliferate by antigen‐nonspecific reagents (mitogens), and it is also critical to study the role of these mitogens in B cell responses. Both of these systems have the advantage that the majority of B cells will be activated. The first basic protocol describes B cell proliferation induced by two commonly used stimulants–anti‐Ig antibody (either anti‐IgM or anti‐IgD) and lipopolysaccharide (LPS)–as measured by incorporation of [3H]thymidine into dividing cells. Alternate protocols describe other commonly used mitogens as well as other means of measuring cell proliferation.

IL4 is not required for proliferative responses in B cells stimulated by anti-IgD-dextran conjugates even under limiting conditions of cell-cell interaction

The experiments in this manuscript confirm and extend our previous finding that IL4 has minimal enhancing activity on B cell activation stimulated by anti-Ig-dextran conjugates. The absence of significant IL4-mediated enhancement is seen also under conditions which are limiting for optimal B cell proliferation. Thus, even when B cells are cultured at low cell densities where cell to cell contact is minimized and are stimulated with picogram per milliliter concentrations of anti-Ig-dextran, IL4 mediates low levels of enhanced proliferation, if at all. The low level of IL4-induced enhancement does not reflect the anti-Ig-dextran-mediated downregulation of IL4 receptors on B cells, since anti-Ig-dextran stimulates an increase in IL4 receptors similar in magnitude to that stimulated by IL4 by itself. To exclude the possibility that anti-Ig-dextran was stimulating IL4 secretion by B cells and thus masking an effect of added IL4, we added inhibiting concentrations of monoclonal anti-IL4 antibody, with the B cells and found that it was without effect on anti-Ig-dextran-stimulated proliferation. Our results suggest that IL4 may not have a prominent role in influencing B cell growth that is stimulated by multivalent T cell-independent antigens.

In Vitro Antibody Production

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.