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

• Published in: Biotechnology and bioengineering Vol. 50; no. 5; pp. 529 - 540
• Main Authors: Peshwa, Madhusudan V., Page, Laura A., Qian, Lichuan, Yang, Demao, van Schooten, Wim C. A.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05.06.1996
• Subjects: CTL; dendritic cell; HTLV-1; immunotherapy; T-lymphocytes
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19960605)50:5<529::AID-BIT7>3.0.CO;2-H

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.