Cytotoxic T-Lymphocytes Recognize and Target Leukemic Stem Cells

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Treatment for acute myeloid leukemia (AML) patients became more effective, yet relapse rates are still high. Immunotherapeutic approaches might overcome the immune escape mechanisms of LSC and might be a therapeutic option. Leukemia-associated antigens (LAA) represent targets that are recognized by cytotoxic T-lymphocytes (CTL) and might be useful for specific immunotherapy.

We enriched the LSC containing fraction as CD34+CD38- cells (called LSC-EF) of 20 AML patients using FACS, analyzed expression patterns of LAA in LSC-EF and compared them to patterns in enriched HSC und bulk AML cells by microarray expression analysis and qRT-PCR. In immunoassays, we analysed the functional inhibition of LCS in colony forming assays (CFU) of AML patients by stimulated T cells.

We detected a high expression of several LAA in LSC-EF. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC-EF compared to HSC. Survivin (p = 0.7), HAGE (p = 0.5), SSXIP2 (p = 0.4) and Aurorakinase A (p = 0.7) did not indicate a significant differential expression. BAGE does not seem to be an ideal candidate as it is significantly higher expressed on normal HSC (p = 0.0056). Further, we evaluated the expression differences of LSC-EF compared to leukemic bulk. PRAME, RHAMM and WT1 qualified as interesting potential targets as they are lower expressed on leukemic bulk than on LSC-EF. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk but lower in LSC-EF. qRT-PCR confirmed our array data.

In immunoassays, T cells stimulated against LAA indicated a significant inhibition of CFUs in AML patient samples. PRAME, RHAMM and WT1 showed good immunogenic responses with CTL recognition rates in a range of 58-83%. PRAME hereby proved as most effective using two different patient samples, with a reduction of 83% colonies (p = 0.004). WT1 shows a significant reduction of 74% (p = 0.05) and RHAMM of 58% colonies (p = 0.2).

Taken together, several LAA are significantly expressed on the LSC-EF, whereas PRAME, RHAMM and WT1 seemed most promising as these LAA are differentially expressed compared to normal stem cells and stimulation of cytotoxic T cells against these LAAs inhibit LSC in CFU assays.