Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.

Abstract

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure