Cellular Immunotherapy of Acute Leukemias After High Dose Chemotherapy with Cytarabin (ARA-C) and Cyclophosphamide (CY) in a Murine Model

Abstract

Allogeneic lymphocytes are able to eradicate resistant leukemia cells after bone marrow transplantation. We developed a murine model to investigate their effectiveness for the prevention of leukemia relapse after high dose chemotherapy. Method: 1 × 105 A20 leukemia cells (H-2^d, B cell neoplasm) were injected into Balb/c (H-2^d) mice. Five days later, animals were treated with ARA-C (2 × 150 mg/kg i.p. daily; day 1–3) which provides high antileukemic activity against A20. To ensure sufficient immunosuppression, increasing doses of CY (60 to 125 mg/kg i.p.) were additionally given at day 4+5. At day 8 and 11, 2 — 107 either allogeneic MHC-mismatched (C57Bl/6, H-2^b) or allogeneic, MHC-matched (DBA, H-2^d) spleen cells were injected intravenously. Leukocyte counts were determined every three days. Leukemia relapse was denned as death with spleen weight > 0.15g and visible tumor nodules. Results: Without further therapy, relapse free survival was 0% with a median survival time (MST) of 21 days. Treatment with ARA-C and 2 × 60, 2 × 100, or 2 × 125 mg/kg CY cured 29%, 38% and 75% of the animals (MST = 45, 56, >200 days), respectively. After additional injection of MHC-mismatched allogeneic spleen cells, similar leukemia free survival (LFS) rates were observed: 38%, 45%, and 86% (MST = 52, 47, >200 days). Analysis of leukocyte counts revealed that 2 × 125 mg/kg CY were necessary to induce severe lymphocytopenia (<500/nl) in the majority of animals. Thus, at least in the groups treated with lower doses of CY, our negative findings could be explained by insufficient immunosuppression of the recipient (resulting in rapid rejection of allogeneic effector cells). When cells with a lower immunogenetic barrier to the recipient were used (DBA) after immunosuppression with 2 × 60 mg CY, a significant reduction of the relapse rate from 71% to 56% was observed. Conclusions: Our preclinical model allows the investigation of cellular immunotherapy after chemotherapy. Future studies will focus on the development of preparative regimens with better immunosuppressive potential. The separation and stimulation of allogeneic NK cells with antileukemia activity will enable us to transfer large effector cell numbers without GvHR.