Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

Abstract

CD8+ cytotoxic T‐lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide‐specific CD8+ T‐lymphocytes from the peripheral blood of healthy, human trophic leukemia virus‐1 (HTLV‐1) seronegative human leucocyte antigen (HLA)‐A*0201 individuals. A combination of density‐based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen‐presenting cells (APCs), and T‐lymphocytes. The DCs were pulsed with HLA‐A*0201 binding peptides and cultured with autologous T‐lymphocytes to generate peptide‐specific CTLs. The CTLs were generated against a nine‐amino‐acid peptide from the Tax protein of HTLV‐1. The CTLs were expanded according to a restimulation schedule employing peptide‐pulsed autologous monocytes and low‐dose interleukin‐2 (IL‐2) to numbers in excess of 100 × 106 cells following 5 weeks of culture. Expanded cells contained primarily CD3+ T‐cells, of which CD8+ T‐lymphocytes constituted greater than two‐thirds of the cell population. Obtained CTLs exhibited potent antigen‐specific lysis of peptide‐pulsed target cells in a dose‐dependent fashion in in vitro 51Cr release cytotoxicity assay. This antigen‐specific killing was shown to be HLA class I restricted and mediated by CD8+ T‐lymphocytes. Since the T‐lymphocytes were obtained from HTLV‐1 seronegative donors, the generation of peptide‐specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. © 1996 John Wiley & Sons, Inc.