For effective adoptive cancer immunotherapy, not only killer lymphocytes (cytotoxic T cells or NK cells) but also helper T cells are considered to be beneficial. The conventional culture of human lymphocytes with high-dose of recombinant interleukin -2 (rIL-2) yields a high proportion of killer cells (CD8+ or CD56+ cells) but is usually accompanied by a decrease in the proportion of CD4+ cells. Peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 monoclonal antibody (10 ng/ml) in the absence of rIL-2 during the initial 48-hr period of cultivation followed by the addition of rIL-2 (200 Japan reference units/ml) (referred to as anti-CD3-rIL-2) resulted in CD4+ cells expanding 8.0- to 63.3-fold at day 12-14. When PBMC were cultivated with rIL-2 alone, or with rIL-2 together with anti-CD3 initially, CD4+ cells expanded 1.7-6.2- or 5.7-36.9-fold, respectively. The expansion of CD4+ cells from PBMC in the anti-CD3-rIL-2 cultures was enhanced by the addition of anti-CD28 monoclonal antibody at the initiation of cultures (500 ng/ml) (referred to as anti-CD3/anti-CD28-rIL-2) (12.1- to 127.8-fold expansion). The anti-CD3-rIL-2 and anti-CD3/anti-CD28-rIL-2 cultures also demonstrated a lesser expansion of CD3-CD56+ cells (NK cells). The PBMC in the anti-CD3/anti-CD28-rIL-2 cultures showed low cytotoxic activity per cell; however, the cytotoxic activity per total culture was comparable to that of PBMC expanded with rIL-2 alone because of the larger total expansion of the former culture.(ABSTRACT TRUNCATED AT 250 WORDS)