Generation of dendritic cells from CD34+ cells from patients with acute lymphocytic leukaemia

Abstract

Childhood acute lymphocytic leukaemia (ALL) is highly responsive to chemotherapy and ∼70% of children are cured. However, children with persistent minimal residual disease are high risk for relapse post-transplantation and may require more effective treatment strategies. Adoptive immunotherapy, using anti-leukaemic cytotoxic T-lymphocytes, may improve survival in these patients. Dendritic cells (DC) are professional antigen presenting cells which play an essential role in induction of immune responses. In this investigation, we have attempted to generate DC from ALL samples at presentation and from samples taken prior to bone marrow transplantation (BMT). Mononuclear BM cells from 5 newly diagnosed ALL patients and 22 patients in clinical remission were enriched for CD34+ cells by immunomagnetic selection. CD34+ cells were expanded in serum free medium (SFM) supplemented with Flt3 ligand, G-CSF, GM-CSF, IL-3, IL-6, and SCF for 7 days. Subsequently, cells were cultured for 14 days in SFM supplemented with Flt3 Ligand, GM-CSF and TGFβ. In presentation cases, IL-1β and IL-7 were also added to promote growth of lymphoid DC. Cells were then analysed by flow cytometry for expression of DC-associated antigens. There was a 3 log increase in the proportion of ALL blast cells which expressed the DC-associated antigens CD1A/CD83 following the culture period. These CD1A+/CD83+ cells also expressed CD80, CD86 and HLA-DR. The majority of these cells (65-88%) retained their expression of CD34, CD10 and CD19 suggesting that the DC were of lymphoid origin. A 2-3 log increase in the proportion of cells expressing CD1A/CD83 was obtained using the 22 pre-BMT samples; these cells also co-expressed CD80, CD86. To date, significant antigen specific proliferation of CD8+ T cells has been induced with HLA-A2 pulsed DC. This was verified using tetrameric complexes of HLA class I/antigenic peptide. These findings suggest that functional DC can be generated from cells of ALL patients at presentation and in remission. It may be possible to utilise these ex vivo generated DC for immunotherapeutic strategies in ALL.