Activation of T and B Lymphocytes in Vitro

Abstract

Abstract Biochemical and physicochemical analyses were made of allogeneic effect factor (AEF) prepared in either serum-containing or serum-free mixed lymphocyte cultures of alloantigen-activated T cells and the appropriate target cell population. Ion-exchange chromatography of Sephadex G-100 prepurified AEF on DEAE-cellulose indicated that the active molecules in AEF are relatively homogeneous and slightly negative in charge. Affinity chromatography of AEF on concanavalin A-Sepharose demonstrated that AEF contains glycoprotein, since the bulk of AEF activity was retained on such columns and could be recovered by elution with methyl-α-d-glucopyranoside. Analysis of AEF by dissociative chromatography in 6 M guanidinium-HCl demonstrated a subunit composition consisting of one heavy component of approximately 40,000 daltons and one light component of approximately 10,000 to 12,000 daltons associated noncovalently. Neither of these subunits, derived from dissociative chromatography of serum-containing AEF, alone was capable of reconstituting responses to sheep erythrocytes of T cell-depleted spleen cell cultures; however, addition of the two components simultaneously to such cultures resulted in recovery of full AEF activity. Dissociative chormatography of serum-free AEF yielded similar fractions, although the light component alone in such preparations exerted substantial AEF-like activity. Nevertheless, synergistic effects in terms of reconstituting activity were clearly observed when appropriate concentrations of heavy and light subunits from serum-free AEF were recombined in the same cultures.