Generation of high quantities of viral and tumor-specific human CD4+and CD8+T-cell clones using peptide pulsed mature dendritic cells

CD4 + and CD8 + T cells are key components of immune response against tumor s and viruses Many techniques have been used to clone and expand these cell s in vitro for purposes of immunotherapy. Here, we describe an improved met hod to obtain large quantities of tumor and virus-specific human CD4 + and CD8 + T-cell clones. T cells derived from peripheral blood mononuclear cell s (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detect ed by IFN-gamma intracellular staining and were cloned by limiting dilution . Specific polyclonal T-cell populations were derived for all epitopes pres ented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure , it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore , clones could be maintained in culture in the presence of IL-2 for at leas t I month without losing their antigen-specific reactivity (e.g. cytokine s ecretion, cytolytic activity and proliferation). Importantly, a majority of the CD8 + T-cell clones recognized endogenously processed antigens. This m ethod is of value for the purposes of adoptive anti-virus or anti-tumor imm unotherapy. (C) 2001 Elsevier Science B.V. All rights reserved.