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Tr1 cells kill primary pediatric acute myeloid leukemia cells
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Figure 1. Pediatric acute myeloid leukemia have three levels of sensitivity to killing by LV-10 cells. (A) Primary pediatric acute myeloid leukemia (pAML) bone marrow aspirates were co-cultured at a 1:1 ratio with LV-10 cells. After 4 days, residual pAML (CD3-) were enumerated by flow cytometry (killing assay). Elimination efficiency (E.E.) was calculated for each LV-10 using the equation 1 - (AML remaining in LV-10 co-culture/AML remaining alone). U937 and K562 cells were included as positive and negative controls, respectively, for killing. The solid line indicates the median elimination efficiency, while the box boundaries indicate the range. Each dot rep- resents the E.E. determined by co-culture with one LV-10 cell line, n=2-4. (B) Left panel contains representative plots of remaining pAML after the killing assay with or without LV-10; numbers on plots indicate the number of pAML cells normalized to the CountBright beads. Gating is set based on pAML cells cultured alone. Right panel graphs the representative examples to show the absolute differences between the number of pAML cells cultured with LV-10 and alone. S: sensitive; IR (or Int. Resistant): intermediate resistant; R: resistant.
without LV-10 cells. Survival of pAML cultured in medium alone did not correlate with their sensitivity to killing when cultured with LV-10 (Online Supplementary Figure S2A). pAML sensitivity to killing also did not correlate with blast percentage within the bone marrow aspirate (Online Supplementary Figure S2B). Notably, pAML sensitivity to killing did not depend on whether the sample was acquired at onset or at relapse. Although our sample set was limited, we observed that six of the seven pAML samples with core- binding factor (CBF) rearrangements (inv(16)(CBFB- MYH11) and t(8;21)(RUNX1-RUNX1T1)), which are asso- ciated with a more favorable prognosis,4 were classified as IR or R.
Killing-sensitive pediatric acute myeloid leukemia (pAML) have significantly different gene expression than resistantpAML
In order to identify factors impacting pAML sensitivity to LV-10 killing, we performed RNA-seq on 14 S, IR, and R pAML. We found 335 differentially expressed genes (DEG) between S and R pAML (absolute log2 fold change [FC] ≥2, false discovery rate [FDR] <0.05) (Figure 2A; Online Supplementary Table S2). Between the other groups, we found 247 DEG between the S and IR pAML, while the IR and R pAML were more similar, with only 27 DEG, (Online Supplementary Figure S3A and B; Online Supplementary Tables S3 and 4).
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