Enhanced Elimination of 6-Mercaptopurine or Methotrexate-Treated Acute Lymphoblastic Leukemia Cells by Natural Killer Lymphocytes In Vitro: A Potential Mechanism of Action of Maintenance Chemotherapy

Maintenance chemotherapy (MC) is an important component of childhood B-precursor acute lymphoblastic leukemia (ALL) therapy; however, it is not necessary in the treatment of mature B cell neoplasms. The operational mechanisms of MC are not understood. Improvement in immunologic function including near normal levels of natural killer (NK) lymphocytes was reported during ALL MC. We hypothesize that in addition to their direct cytotoxicity, MC drugs alter surviving lymphoblasts, rendering them susceptible to innate immune response, likely through cell mediated cytotoxicity via stress proteins such as NKG2D ligands, co-stimulatory or adhesion molecules.

The effect of 6-mercaptopurine (6MP) or methotrexate (MTX) treatment of B-precursor and mature B leukemia/lymphoma cells in their elimination by NK lymphocytes was investigated in this study.

Allogeneic NK cell-mediated elimination of REH (TEL/AML-positive B-precursor ALL) and Raji (mature B cell lymphoma) cells treated with standard MC drugs was studied. High dose cytarabine (Ara-C) and MTX are used during the consolidation chemotherapy; therefore, Ara-C and MTX-resistant REH and Raji cell sub-lines were established by exposing wild type cells to increasing concentrations of drugs over several months. Natural killer cells from 17 healthy volunteers were separated using the MACS NK cell isolation kit. After purity evaluation, NK cells were incubated with interleukin-15 overnight. Leukemia cells were incubated in minimally toxic (20% cytotoxicity) concentrations of 6MP and MTX. The leukemia/lymphoma cells were then co-incubated with NK cells at different ratios. The NK cell-mediated leukemia/lymphoma cell cytotoxicity was measured by flow cytometric cell-mediated cytotoxicity assay, marking effector cells with lineage-specific monoclonal antibodies and staining target cells with propidium iodide and annexin-V and using microspheres for quantification of viable and apoptotic cells. The level of resistance of the respective cell sub-lines was evaluated using MTT assay. We also investigated whether NK cell exposure to same concentrations of MC drugs before co-incubation alters cytotoxicity. Surface expression of NKG2D ligands, ULBP 1, 2 and 3, MICA and MICB was studied by flow cytometry.

6-mercaptopurine treatment of REH cells and MTX treatment of Raji cells resulted in enhanced NK cell-mediated elimination when compared to untreated cells by 25% and 20%, respectively. The results were similar when NK cells were exposed to the same concentrations of MC drugs before co-incubation, indicating lack of negative effect of the drug exposure in NK cells’ ability to kill. Similar experiments were conducted on resistant cells, in order to make the target cells more comparable to the residual lymphoblasts during MC. Most interestingly, the REH cells, but not the Raji cells, resistant to Ara-C and MTX showed about 14% and 4% enhancement of NK cell-mediated killing, respectively, after being exposed to the minimally toxic concentrations of MC drugs. This indicates that resistant B precursor ALL cells can be eliminated by NK cells upon MC drug exposure, but not mature B lymphoblasts, in this experimental setting. No increase in the expression of NKG2D ligands on drug treated ALL cells was observed.

These findings suggest that enhanced susceptibility of drug-exposed leukemia cells to innate immune response may be an operational mechanism of MC. This mechanism may involve pathways other than NKG2D.

No relevant conflicts of interest to declare.