The preferential expansion of functional CD4+ lymphocyte populations in vitro

We describe a simple and inexpensive chemical procedure for the selective expansion of human CD4+ lymphocytes. The method employs L-leucine methyl ester (LME) to deplete monocytes and large granular lymphocytes, as well as to inhibit growth of CD8+ lymphocytes. LME treatment eliminates granular cells, but most CD8+ lymphocytes, B-lymphocytes, and CD4+ lymphocytes remain. Peripheral blood mononuclear cells (PBMCs) from normal and HIV-positive individuals are treated with LME for 1 h at ambient temperature and cultured in the presence of IL-2 to expand the cell number. Stimulation with the T-cell mitogens concanavalin A, phytohemagglutinin, or OKT3 antibody augments lymphocyte expansion and within 1-3 weeks the culture is greatly enriched (90-100%) in CD4+ lymphocytes. LME-treated lymphocytes expand up to 10-fold during culture in the presence of IL-2 alone and up to 400-fold following treatment with T-cell mitogens. The immune function of LME-treated and expanded peripheral blood lymphocytes was examined using the response to the recall antigens tetanus toxoid and Candida albicans. Fresh PBMCs exposed to these recall antigens proliferated readily. Similarly, LME-treated lymphocytes following expansion responded to these recall antigens with good fidelity to the original PBMC response patterns in four of six donors. The expanded and LME-treated lymphocytes also exhibited good mitogen responses in three of three donors. The LME procedure allows for the simple and inexpensive generation of expanded, immunologically functional, CD4+ lymphocytes.