Assembling the Players for Evaluation of Anti-Leukemic CTL Activity in NOD-SCID Mice.

Acute Lymphocytic Leukemia (ALL) patients, who relapse after hematopoietic stem cell transplantation, have a poor prognosis with few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect post transplant may improve the survival of relapsed patients. We postulate that dendritic cells (DC) derived from cord blood CD34⁺ stem cells will be ideal for this. Further we propose the use of RNA as a source of antigen to induce T cell responses to poorly immunogenic tumours, such as ALL. We have shown that CD34⁺ cells can expand and differentiate into DC and are functional antigen presenting cells. In addition, we regularly achieve >90% transfection efficiency with >90% cell viability when CD34⁺-DC are electroporated with eGFP mRNA. Electroporation of CD34⁺-DC with flu mRNA results in processing, translation and presentation of epitopes by the DC, which are recognised by flu-specific cytotoxic T lymphocytes (CTL). We have induced anti-leukaemic CTL with leukaemic total RNA from the HLA-A2⁺ NALM-6 cell line. We have established a NOD-SCID mouse model of primary human ALL to test the efficacy of the anti-leukaemic CTL in vivo. Mice engraft within 4–6 weeks of transplantation. Mice transplanted with ALL3 had 93.9±0.8% (BM) and 84.3±4.7% (spleen) CD45⁺cells at sacrifice. Mice transplanted with ALL7 had 79.1±5.5% and 89.9±1.2% CD45⁺ cells in the BM and spleen respectively. Cells from the BM and spleen have been cryopreserved and total RNA extracted for later CTL generation. Experiments aimed at replicating the minimal leukaemia burden post transplant, show that the kinetics of appearance of human cells in the blood and the time of palpable splenic engraftment relates to the cell dose. This suggests that while the leukaemic burden is low, the kinetics of growth are identical and thus, by varying the inocula we model emerging relapse. Thus, we have “assembled the players” and are ready to test the efficacy of anti-leukaemic CTL generated by stimulation with RNA loaded CD34⁺-DC to control in vivo leukaemic growth.