B cell proliferation and immunoglobulin production by supernatants from Nef-expressing macrophages requires the presence of ferritin. (A) B cell proliferation depends on the presence of ferritin. Supernatants from macrophages expressing HIV-1 Nef, GFP, or untreated macrophages were immunodepleted with ferritin-specific or isotype antibodies (left hand panel) then examined for induction of proliferation in cultures of purified B cells (right hand panel). (B) Ferritin induces the production of immunoglobulin from B cells. Supernatants from Nef-expressing, GFP-expressing or mock-treated macrophages were immunodepleted by ferritin-specific or isotype antibodies as in (A), and assayed for the efficiency of ferritin immunodepletion (left hand panel) and for their ability to induce immunoglobulin production from purified B cells (right hand panels). (C) Nef immunodepletion does not impact B cell stimulation by supernatants of Nef-expression macrophages. Supernatants of Nef or GFP-expressing macrophages or mock-infected macrophages were treated with three Nef monoclonal antibodies or isotype controls and assayed for the ability to induced B cell proliferation. (D) Supernatants from macrophages treated with recombinant HIV-1 Nef or MA proteins do not induce B cell proliferation. Macrophages were treated with the recombinant proteins over a 6 log range and subsequently assessed for ability to induce B cell proliferation.