Influence of antigen presentation and exogenous cytokine activity during in vitro primary immunizations employed for the generation of monoclonal antibodies.

Hybridomas secreting monoclonal antibodies (MAbs) against African horse sickness virus (AHSV) were generated using different AHSV antigen preparations (inactivated AHSV, semi-purified virus, and a preparation of nonstructural viral proteins) in one of three different in vitro primary immunization systems: (i) the Cel-prime kit, a method using immunization of splenocytes aided by antigen-primed support cells; (ii) a system based on a cytokine soup derived from a mixed lymphocyte reaction plus stimulated EL4-IL-2 cells; (iii) a system based on a cytokine soup derived from splenocytes stimulated by pokeweed mitogen in order to obtain a mixture of cytokines enriched for Th2 lymphokines. The viability of immunized BALB/c mouse splenocytes, immunoglobulin production by the subsequently generated hybridomas, and the specificity of the MAbs were compared. The most efficient in vitro primary immunization system was the Cel-prime system employing semi-purified antigen. This efficiency was manifest in terms of a greater viability of the splenocytes in the immunization, as well as a higher number of specific antibody-secreting hybridomas. It seems probable that the support cells of the Cel-prime system have an accessory function such as that attributed to antigen-presenting cells. Such a function would result in impairment of apoptosis, and thus increase the viability of the splenocytes in the in vitro primary immunization system, as well as enhancing stimulation of the immune response against the antigen used. The presence of cytokines at the beginning of the in vitro primary immunization did have an influence, but this was secondary to what appeared to be the major event of cellular interaction associated with the accessory cell function of the support cells.