Characterization of a Db-specific helper factor required for the induction of cytotoxic responses to alloantigens with the use of monoclonal antibodies specific for the helper factor or the T-cell antigen receptor

Abstract

A T helper clone (clone 9), isolated from a H-2d anti-H-2b mixed lymphocyte culture, was previously found to produce an antigen-specific helper factor (ASHF) that could be specifically absorbed out with BIO.A(2R) (KkAkEkDb), but not B10.A (KkAkEkDd), spleen cells. In order to characterize this ASHF further, we have constructed T-cell hybridoma lines by fusing clone 9 cells with the AKR thymoma, BW5147. One of these hybridoma clones, referred to as clone 25, produced an ASHF that was specific for the Db alloantigen. Immunization of allogeneic C57BL/6 mice with clone 9 cells and subsequent fusion of these immune spleen cells with non-secreting myeloma cells led to the isolation of a monoclonal antibody (mAb) (clone 30 IgM) that was capable of neutralizing the helper activity of clone 25 ASHF. Clone 30 IgM affinity column was found to retain clone 25 ASHF; clone 30 IgM column eluates augmented the cytotoxic responses of CBA/J thymocytes to B6(H-2b), but not D2(H-2d), alloantigens. Preabsorption of clone 25 ASHF with Db-bearing spleen cells prior to affinity purification over a clone 30 IgM column resulted in the abrogation of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band in SDS-polyacrylamide gels run under reducing conditions. Clone 25 ASHF was also retained by immunoadsorbents made with an IgG2a mAb (F23.1) the reactivity of which is against the beta chain of the T-cell receptor. Furthermore, affinity purification of clone 25 ASHF over a F23.1 affinity column, but not an irrelevant mAb column, also yielded a 50,000 MW molecule. These findings suggest that this particular ASHF may be intimately related to the T-cell antigen receptor.